novel antiproliferative factor and methods of use

ABSTRACT

A novel antiproliferative factor comprising a glycopeptide is disclosed. In specific embodiments, the novel antiproliferative factor is associated with the bladder. Compositions, diagnostic kits and reagents, and methods of using the compounds for identifying and/or treating interstitial cystitis and cancer are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/484,010, filed Jul. 1, 2003; U.S. Provisional PatentApplication Ser. No. 60/515,850, filed Oct. 29, 2003; and U.S.Provisional Patent Application Ser. No. 60/569,363, filed May 7, 2004,all of which are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was generated at least in part with funds fromNational Institutes of Health NIH, NIDDK DK52596; the VeteransAdministration (VA Merit Review Funding); and intramural National CancerInstitute funds. The United States Government may have certain rights inthe invention.

FIELD OF THE INVENTION

The present invention is directed to fields of medicine, biochemistry,cell biology, and chemistry. More specifically, the present inventionaddresses a novel compound and derivatives thereof having growthinhibitory activity. The present invention is further directed to usesof the novel compounds and derivatives thereof as a biomarker and/or adiagnostic for bladder disorders, particularly interstitial cystitis.The present invention also relates to the treatment of any diseaseinvolving uncontrolled cell proliferation, such as cancer.

BACKGROUND OF THE INVENTION

Approximately one million people in the United States suffer from thebladder disorder interstitial cystitis, which is a chronic painfulurinary bladder condition characterized by thinning or ulceration of thebladder epithelial lining (Curhan et al., 1999).

Cystoscopic abnormalities seen in the bladder of patients with thisdisorder include petechial hemorrhages called “glomerulations” andulcers that extend into the lamina propria (Hunner's ulcers) (Johanssonand Fall, 1990; Skoluda et al., 1974). The most consistent histologicabnormalities include denudation or thinning of the bladder epitheliumto 1-2 cell layers (Johansson and Fall, 1990; Skoluda et al., 1974;Tomaszewski et al., 2001). These findings suggest that interstitialcystitis may be caused by an inhibition of normal bladder epithelialcell proliferation, resulting in a loss of epithelial barrier integritywith subsequent exposure of sensory nerve cells in the bladder wall tourinary constituents. However, the pathogenesis of interstitial cystitisis heretofore unknown.

The isolation of an antiproliferative factor (“APF”) peptide that ismade uniquely by bladder epithelial cells from interstitial cystitispatients (Keay et al., 2001; Keay et al., 2000) and profoundly inhibitsnormal bladder epithelial cell growth (Keay et al., 2003) was previouslydescribed. U.S. Pat. No. 5,962,645, incorporated by reference herein inits entirety, teaches a purified human antiproliferative factor (APF)isolated from the urine of patients with interstitial cystitis whereinthe APF is characterized by a molecular weight of about 1.7 kDadetermined by mass spectrometry on a sample in an aqueous acetonitrilesolution and a pI range of about 1.38-3.5, and the APF is capable ofinhibiting normal human bladder epithelial (HBE) and bladder carcinomacell proliferation. Picomolar quantities of HPLC-purified APF were ableto induce several changes in normal bladder epithelial cells in vitro,including significantly decreased rates of proliferation (Keay et al,2003) and decreased production of a growth factor required for log-phasegrowth of bladder epithelial cells (heparin-binding epidermal growthfactor-like growth factor, or HB-EGF) (Keay et al., 2000; Keay et al.,2003).

HB-EGF has been previously described (for example, see U.S. Pat. No.5,811,393). U.S. Pat. No. 6,156,522 describes a method for diagnosinginterstitial cystitis in a subject suffering from bladder dysfunction,said method comprising the steps of (a) measuring the levels ofHB-EGF-like growth factor in the urine sample of the subject; and (b)comparing said level with normal levels, wherein decreased levels ofheparin-binding epidermal growth factor-like growth factor, as comparedto levels of heparin-binding epidermal growth factor-like growth factorin a normal population, indicates the presence of interstitial cystitis.U.S. Pat. No. 6,232,289 teaches a method for enhancing bladderepithelial cell proliferation in a subject in need thereof, said methodcomprising administering to the subject HB-EGF, in an amount effectiveto enhance bladder epithelial cell proliferation. U.S. Pat. No.6,376,197 teaches a method for diagnosing a condition such asinterstitial cystitis associated with inhibited bladder epithelial cellproliferation comprising the steps of determining the level of epidermalgrowth factor in urine from the subject; and comparing said level withnormal level, according to the following criterion: increased level ofepidermal growth factor, as compared to level of epidermal growth factorin a normal population, indicates the presence of the condition.

Microarray analysis indicated that APF can also induce changes in thepattern of cellular gene expression toward a more differentiatedphenotype (Keay et al., 2003). Identification of this factor istherefore important for determining its potential role in thepathogenesis of interstitial cystitis and establishing its utility as abiomarker for this disease.

Preliminary characterization of APF indicated that it was a lowmolecular weight, relatively heat stable peptide (Keay et al., 2000).APF is found in minute quantities in both patient urine specimens andexplanted patient bladder cell supernatants, making conventional methodsof structural analysis, such as NMR spectroscopy, unfeasible. Asdescribed herein, the complete characterization of this potent growthinhibitor provides a novel structure not previously disclosed. Thecomplete characterization was made using a combination of techniquesincluding mass spectrometry, lectin affinity chromatography, enzymaticanalysis, and total synthesis.

Confirmation of the structure of APF was provided using microcapillaryliquid chromatography of native and synthetic APF derivatives, as wellas by demonstration of synthetic APF's ability to regulate growth factorproduction and bladder epithelial cell proliferation. Additionalevidence of APF's identity was provided by identification of mRNA thatbound to a probe for the frizzled 8-protein segment in cells frominterstitial cystitis patients but not controls, as well as by bindingof rabbit antibodies raised against synthetic APF to purified native APFfrom supernatants of bladder epithelial cells of interstitial cystitispatients.

Another factor considered to be a cell cycling inhibitory factor isdescribed in U.S. Pat. No. 5,916,871, wherein the factor comprises asialylated glycopeptide preferably having a molecular weight much larger(e.g. 18 kD or 66 kD) than APF and that inhibits the G1 phase (but notG2) of the cell cycle. This previously described factor also had acarbohydrate component that accounted for less than 10% of its totalmass, whereas APF in its native form has carbohydrate that accounts for44% of its total mass.

Other and further objects, features, and advantages will be apparentfrom the following description of the presently preferred embodiments ofthe invention, which are given for the purpose of disclosure.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a system and method that concern anantiproliferative factor (APF). In particular aspects of the invention,the APF is associated with, related to, localized in, or otherwiseprovided in vitro; in vivo in an organism, such as in a mammal,including a human; and/or ex vivo, as in a particular tissue, fluid, ororgan. It is also contemplated that APF is provided by lower life forms,such as Drosophila, because it bears homology to a segment of a proteinfound in animals such as humans, mice, and Drosophila. The APF may beassociated with a particular tissue, fluid, or organ from an organism.In specific embodiments, the APF is secreted by cells in the particulartissue, fluid, or organ, and in additional embodiments the APF is activein the particular tissue, fluid, or organ, such as being active in cellscomprised therein. In some embodiments the APF is secreted by and activein cells of a particular tissue, fluid, or organ. In a particularaspect, the APF is associated with tissues comprising epithelial cells,including the following: urinary bladder; ureter; urethra; lung; heart;gastrointestinal tract (including the stomach, small intestine, largeintestine, rectum, liver, pancreas and gall bladder); spleen; malereproductive tract, including the seminal vesicles, prostate,bulbourethral gland, vas deferens, epididymis, testes, and penis; femalereproductive tract, including the ovaries, Fallopian tubes, uterus,cervix, and vagina; kidneys; adrenal glands; thymus; thyroid; skin; bone(including synovium); ocular tissues (including cornea, retina, andlens); cochlea; breast tissue; lymph nodes; oral mucosa (includinggingival), salivary gland, parotid gland; and nasopharygeal mucosa(including sinus mucosa), for example.

In certain embodiments of the present invention, the inventive compoundcomprises an isolated APF molecule. The APF molecule of the presentinvention comprises a glycopeptide that inhibits in part or in full cellproliferation and/or slows cell proliferation, for example. In specificembodiments, the cell being proliferated is an epithelial cell. Inparticular embodiments, APF may be considered a negative growth factor,negative growth regulator, or toxin. Although in specific embodimentsthe APF is a urinary bladder-specific APF or a urinary bladderepithelial cell-specific APF, it is also useful for other tissues, suchas to inhibit the proliferation of non-bladder cells, includingfibroblasts or prostate cells, for example. Because interstitialcystitis patients have the same alterations in the serum levels ofHB-EGF and EGF in serum as seen in urine specimens (Keay et al., 2000),APF in some embodiments is produced in tissues other the urinarybladder.

As delineated in Rashid et al. (2004), in specific embodiments APF is acell-cycle modulator. That is, explanted cells from normal bladderbiopsy specimens were exposed to APF, which increased significantly theproportion of tetraploid and hypertetraploid cells compared to controls.Thus, in a particular aspect of the invention, exposure of a cell to APFprovides a block in cell cycling in the G2 and/or M phase cell cycleblock and/or the production of polyploidy. As such, APF affects cellcycle distribution, which in particular embodiments contributes at leastin part to the pathogenesis of bladder disorders such as interstitialcystitis, for example through disruption of normal urothelialproliferation and repair processes. In further embodiments, exposure ofone or more cells to APF results in inhibition of proliferation of theone or more cells, which may comprise a cell cycle block at any point inthe cell cycle, although in particular embodiments the block isprimarily in G2 or M phase. In particular embodiments, removal of APFpermits the cell cycle to resume.

In particular embodiments, the molecular weight of APF is less thanabout 3000 Daltons, although in alternative embodiment the molecularweight of APF is more than about 3000 Daltons. The composition may befurther defined as comprising a sugar moiety and a hydrophobic moiety,wherein the hydrophobic moiety may be a peptide moiety or a lipidmoiety. Particular peptide moieties include any suitable structure,although in specific embodiments they may be linear, cyclical, branched,or a combination thereof, for example. In further specific embodiments,the peptide moiety comprises homology to at least part of a frizzledpolypeptide, such as having homology to at least part of a transmembranedomain of frizzled 8, such as one comprised in the exemplary sequencedescribed in NP_(—)114072 (SEQ ID NO:7), encoded by the exemplarypolynucleotide in NM_(—)031866 (SEQ ID NO:8). In other specificembodiments, the peptide component of APF comprises total orsubstantially total homology to at least part of the putative sixthtransmembrane domain of frizzled 8, a G-protein coupled receptor whosenatural ligand is Wnt, an important regulator of cell proliferation. Anexample of a secreted frizzled related protein is described in U.S. Pat.No. 6,600,018, which is incorporated by reference herein in itsentirety.

For example, the peptide moiety may comprise less than about 50%, about50% homology to at least part of frizzled 8, about 55% homology, about60% homology, about 65% homology, about 70% homology, about 75%homology, about 80% homology, about 85% homology, about 90% homology,about 95% homology, or 100% homology. A skilled artisan is aware,however, that in those embodiments involving, for example, peptidemimetics sequence homology is not used to determine functionality, butrather chemical characteristics of hydrophobicity and physical andchemical similarities (i.e. polarity, steric bulk, hydrogen bodingcapabilities).

In an object of the invention, APF is a sensitive and specific biomarkerfor a bladder disorder such as interstitial cystitis (IC), and in aspecific embodiment it plays a critical role in the pathogenesis of ICby profoundly inhibiting bladder cell proliferation, such as viaregulation of gene expression (such as by increased E-cadherinproduction, for example) and alterations in the production of specificgrowth factors (such as HB-EGF and EGF, for example). More specifically,the inhibition of bladder cell proliferation by APF involves inhibitionof HB-EGF production; stimulation of cellular EGF, E-cadherin,arylsulfatase A, phosphoribosylpyrophosphate synthetase-associatedprotein 39, or SWI/SNF complex 170 kDa subunit gene expression; orinhibition of cellular putative tRNA synthetase-like protein, vimentin,neutral amino acid transporter B, possible GTP-binding protein, alpha 1catenin, alpha 2 integrin, cyclin D1 and JNK or ribosomal protein L27agene expression.

In one specific aspect of the invention, APF is an acidic, heat stablesialoglycopeptide comprising 9 amino acid residues (such as, forexample, TVPAAVVVA, SEQ ID NO:1; SVPAAVVVA, SEQ ID NO:3; TVPAAVVLA, SEQID NO:4; or SLPAAVVVA, SEQ ID NO:5) covalently linked through theN-terminal threonine, serine, or cysteine, for example, to anN-acetylgalactosamine or N-acetylglucosamine residue that is linked viaan α or β configuration to galactose, and sialylated on the galactosemoiety via 2,3 linkage. The anomeric configuration of the glycosyl bondis alpha in particular embodiments, although it may be beta inalternative embodiments.

In a specific embodiment of the present invention, the peptide componentof APF is hydrophobic and may be substituted for a non-proteinaceousmoiety that is also hydrophobic, such as a lipid. Thus, in particularaspects of the invention, the peptide/lipid moiety of APF facilitatesassociation with a membrane, such as being inserted, linked, bound,intercalated, or otherwise associated thereto, or, alternatively, bybinding to a membrane surface receptor, and the sugar moiety of nativeAPF comprises a high level of the functional activity of the molecule.

The present invention encompasses isolated naturally-existing APF,synthetic APF, derivatives thereof, or mixtures thereof. Like nativeAPF, synthetic APF profoundly inhibited bladder epithelial cellproliferation (IC₅₀=0.4 nM) and HB-EGF production, while stimulating EGFproduction. Synthetic APF also inhibited proliferation of bladdercarcinoma cells that are sensitive to native APF. In specificembodiments of the invention, desialylated native APF and nonsialylatedsynthetic APF comprise functional activity as their sialylatedcounterparts, although nonglycosylated synthetic peptide and the betaanomer of glycosylated synthetic APF comprised less desirable activity.

In one embodiment, the APF compound comprises a sugar moiety having oneor more sugars, wherein the sugars are referred to herein as a firstsugar, a second sugar, a third sugar, and so forth. Although any of thesugars may be covalently linked to a peptide, for example, in specificembodiments the third sugar is covalently linked to a peptide, such asone having a sequence essentially as set forth in SEQ ID NOS:1, 3, 4, or5, or it may be alternatively linked to a lipid molecule. The sugarmoiety may include naturally-occurring sugars, synthetic sugars,derivatives thereof including sugar mimetic components, and/or anycombination thereof.

Certain compounds of the present invention comprise a peptide moiety,which may be characterized by having a terminal subunit having a polarchemical characteristic and/or a heteroatom therein. The subunits of thepeptide may include naturally-occurring amino acid residues, unnaturalamino acids, derivatives of amino acids, such as methylated amino acids,peptidomimetic components and/or any combination thereof. In alternativeembodiments, the peptide moiety is replaced with a lipid molecule, suchas an omega fatty acid, including myristic or palmitic acid, or otherlipids such as phosphatidylcholine, phosphatidylethanolamine, and thelike.

In preferred embodiments, the sugar molecule includes one or more of asialic acid, galactose, glucose, N-acetylglucosamine, and/orN-acetylgalactosamine, for example. In certain embodiment, the sialicacid molecule is covalently linked to the galactose or glucose through a(2, 3), a (2, 6), a (2, 8), and/or a (2,9) linkage. A skilled artisan isaware of the nomenclature used in sugar/carbohydrate chemistry toidentify the atom at the locations specified. Alternatively, thegalactose or glucose is covalently linked to the N-acetylgalactosamineor N-acetylglucosamine molecule through a 1→3, a 1→6 or a 1→4 linkage.In a further preferred embodiment, the N-acetylgalactosamine orN-acetylglucosamine sugar molecule is linked to the hydrophobic moietyin the alpha configuration.

In those compositions of the present invention comprising a lipidmoiety, the lipid moiety may be a saturated fatty acid such as, forexample, caprylic acid, capric acid, lauric acid, myristic acid,palmitic acid, or stearic acid; an unsaturated fatty acid such as, forexample, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid,and/or arachindonic acid; esterified fatty acids such as, for example,steroid alcohols in which an ester is added to an alcohol on the lipid;omega fatty acids, including myristic and palmitic acids;phosphate-containing fats such as, for example, phosphatidylcholine andphosphatidylethanolamine; or surfactants characterized by having a polarhead and a non-polar tail. In general, the critical property of thelipid is the chemical characteristic of hydrophobicity such that thelipid is able to associate, intercalate or otherwise bind to a bilayerlipid cell membrane.

Also contemplated are derivatives of APF in which the peptide having asequence essentially as set forth in SEQ ID NO:1 is a fragment thereof,wherein the fragment is 1 to about 8 amino acids of SEQ ID NO:1.Analogous fragments are contemplated for SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, and SEQ ID NO:6. It is further contemplated that the peptidemoiety may be a nonapeptide or longer than a nonapeptide, such as having10 or more amino acids in the peptide moiety, or having 15 or more aminoacids in the peptide moiety.

It is contemplated that among the derivatives of APF are naturalprecursors or metabolites of APF. Further, natural or synthetic APF ortheir derivatives may be labeled with a detectable molecule such as, forexample, a fluorescent, colorimetric, or radioactive moiety. In allcases, the compounds of the present invention alter cellular functionsin a manner similar to or identical to the alterations affected bynative APF. Examples of cellular functions altered by APF includeinhibition of HB-EGF production; stimulation of cellular EGF,E-cadherin, arylsulfatase A, phosphoribosylpyrophosphatesynthetase-associated protein 39, or SWI/SNF complex 170 kDa subunitgene expression; or inhibition of cellular putative tRNA synthetase-likeprotein, vimentin, neutral amino acid transporter B, possibleGTP-binding protein, alpha 1 catenin, alpha 2 integrin, cyclin D1 andJNK or ribosomal protein L27a gene expression.

Oligonucleotides that encode the nonapeptide of SEQ ID NO:1 orbiologically functional erivatives thereof, such as the peptides of SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5, for example, and/or precursors ofAPF are also contemplated herein. An exemplary oligonucleotide thatencodes SEQ ID NO:1 is SEQ ID NO:2. However, given the limited choicesof triplet nucleotides per given codon for a particular amino acid, askilled artisan recognizes that a polynucleotide encoding a peptide ofthe invention, exemplary embodiments of which include SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 are limited in numberand are well within the scope of the invention. This is particularlytrue given the further subset of codons available for encodinghydrophobic amino acids, which are preferably comprised in at least partof the peptide moiety of the APF molecule.

Compositions comprising the APF compounds of the present invention arecontemplated. The compositions may further comprise a delivery agent,such as a liposome; encapsulated cell; conjugated molecules, such asantibodies (Safavy et al., 2003), other peptides, and a variety ofnon-peptide conjugates (including folate and polyethylene glycol; Aronovet al., 2003); drugs, such as geldanamycin (Mandler et al., 2004) orinsulin (Ou et al., 2003); liposomes (Heath and Martin, 1986);lactosaminated human albumin (Di Stefano et al., 2003); polyethyleneglycol (PEG) (Aronov et al., 2003); nanoparticles, such as colloidalgold; or other molecules that bind to cell surface receptors tofacilitate cellular interaction with or uptake of APF.

Use of such novel compositions is another object of the presentinvention. Specifically, the present invention is directed to adiagnostic kit for the detection of a bladder disorder, morespecifically interstitial cystitis, comprising a compound of the presentinvention. In specific embodiments, a diagnostic kit of the presentinvention comprises one or more antibodies to an epitope on the peptide,sugar, or glycopeptide moiety of an APF molecule. The antibodies may bepolyclonal, monoclonal, or the kit may comprise both polyclonal andmonoclonal antibodies.

In some aspects of the invention, there is a method that detects abladder disorder in an individual by assaying for an APF molecule. Inspecific embodiments, the bladder disorder is interstitial cystitis. Thepresent invention provides the advantage of being able to assay for anAPF molecule by non-invasive means, although in alternative embodimentsinvasive means may be used. A sample may be obtained from the individualand assayed directly or indirectly for the APF molecule. In specificembodiments, the sample comprises urine, plasma, or other bodily fluidspecimen, such as whole blood, feces, saliva, nipple aspirate, mucus, orsweat. A urine sample may be obtained following voiding from theindividual, and it may be obtained via a catheter.

Diagnostic methods may comprise detection of one or more sugar moietiesof the APF molecule and/or they may comprise detection of the peptide orlipid moieties or a combination of peptide and/or lipid and/or sugarmoieties of the APF molecule. In specific embodiments, assaying stepscomprise introduction to a sample an antibody directed to an epitope ofthe peptide moiety of an APF molecule. In embodiments wherein APFcomprises a lipid moiety, this may be used for diagnosing utilizingsuitable assaying means such as standard methods available to detect alipid molecule.

The present invention is also directed to methods of treating epithelialhyperplasia or malignancies of epithelial origin comprisingadministering an effective amount of APF or derivatives thereof to apatient in need of such treatment.

The present invention is directed to methods of treating fibroblasthyperplasia or malignancy comprising administering an effective amountof APF or derivatives thereof to a patient in need of such treatment.

The present invention is directed to methods of treating lymphoreticularmalignancies or solid tumors comprising administering an effectiveamount of APF or derivatives thereof to a patient in need of suchtreatment.

The present invention is directed to methods of treating cancercomprising administering an effective amount of APF or derivativesthereof to a patient in need of such treatment. Any kind of cancer maybe treated, such as bladder, lung, breast, prostate, brain, stomach,colon, spleen, liver, pancreatic, melanoma, head and neck, thyroid, andso forth. In specific embodiments, though, the invention is useful fortreating bladder or prostate cancer, comprising co-administering aneffective amount of APF or derivatives thereof to a patient in need ofsuch treatment. In additional aspects of the invention, the APFimproves, facilitates, or assists in overcoming resistance or improvingsensitivity to a cancer therapy selected from the group of chemotherapy,radiotherapy, surgery gene therapy, and/or immunotherapy.

The compounds of the present invention also have antiangiogenicproperties and are contemplated for use in methods of treatmentsbenefiting from inhibiting or slowing the formation and/ordifferentiation of blood vessels, such as blood vessels that feed atumor.

The APF compounds of the present invention are also useful as anantifungal agent.

The APF compounds may be used to generate antibodies thereto, in whichthe antibodies may be used for treating interstitial cystitis. Inparticular embodiments, the present invention encompasses generatingantibodies against a peptide moiety, sugar moiety, and/or glycopeptidemoiety of an APF composition. For example, antibodies are generatedagainst a peptide moiety of APF, such as one comprising SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, for example. Theantibodies that are generated may be polyclonal or monoclonal. As usedherein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” may also be used to refer to any antibody-likemolecule that has an antigen binding region, and includes antibodyfragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABs),Fv, scFv (single chain Fv), and the like. The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).Antibodies of the invention are prepared by immunizing an animal with acomposition in accordance with the present invention and collectingantisera from that immunized animal.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, or toxins, for example. Adjuvants thatmay be used include IL-1, IL-2, IL-4, IL-7, IL-12, y-interferon, GMCSP,BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP,CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion is also contemplated. MHC antigens may even be used. Exemplary,often preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

Alternatively, the APF compounds are used to prepare antisenseoligonucleotides, small interfering RNA molecules, or other substancesthat inhibit APF production or activity.

In yet another embodiment, the APF compounds are used to generate ananimal model for interstitial cystitis. In an exemplary animal model,the animal is stimulated to develop symptoms (such as bladder or pelvicpain, increased urinary frequency, and/or increased urinary urgency) orfindings (such as thinning and/or ulceration of the bladder epithelium)associated with interstitial cystitis following exposure of the bladderepithelium to APF. This exposure could result from the directadministration of synthetic or native APF intravesically, or theexpression/biosynthesis of APF by bladder epithelial cells followingtransfection with a polynucleotide encoding the peptide moiety of theAPF molecule, for example. The gene can further be introduced into thecells by direct methods (such as electroporation or membrane fusion) orby indirect methods (such as via a viral vector). It should be notedthat the sequence of mouse and human frizzled 8 is identical over the 9amino acids that are homologous to the peptide moiety of the APFmolecule, and mouse fibroblasts are sensitive to the antiproliferativeeffects of APF, allowing for the development of a mouse model for thisdisorder based on the APF. An exemplary human frizzled 8 polypeptide isSEQ ID NO:7, and an exemplary mouse frizzled 8 polypeptide is SEQ IDNO:9 (NP_(—)032084).

In another embodiment, an APF composition is utilized in an animal modelfor psoriasis, such as one comprising spontaneous psoriasiform skinlesions (Schon, 1999). Other exemplary disease models include animalmodels for testing the effects of APF on hyperplasia, including a modelcomprising athymic, asplenic nude rats (Polo et al., 1999) and ahairless guinea pig model for keloid formation (Clugston et al., 1995).

In yet another embodiment, a method of detecting APF activity using acell proliferation inhibition assay (such as inhibition of tritiatedthymidine or bromodeoxyuridine incorporation, for example) using normalbladder epithelial cells or cells derived from bladder carcinomas isprovided, wherein the presence of APF indicates interstitial cystitis ispresent or has the predisposition to develop. These methods areparticularly advantageous, because they may be non-invasive, i.e., thesample may be a urine sample that may or may not be obtained viacatherization. A urine sample may be obtained upon voiding from anindividual.

In another embodiment, there is a method of detecting APF by completinga direct assay for the APF glycopeptide using an antibody-based method(such as ELISA), mass spectrometry (MS) or nuclear magnetic resonance(NMR), or a combination thereof.

The present invention also encompasses APF compositions for use as astimulus, given the ability to stimulate specific cell signalingpathway(s), including (but not limited to) the Wnt signaling pathways.

In an embodiment of the present invention, there is an isolated orsynthesized composition comprising a urinary bladder antiproliferativefactor having one or more sugar moieties, wherein at least one sugarmoiety is linked to a hydrophobic moiety. The composition may be furtherdefined as a urinary bladder epithelial cell antiproliferative factor.The molecular weight of the factor may be less than about 3000 Daltons.In specific embodiments, the hydrophobic moiety comprises one of thefollowing: a peptide moiety, which may be linear, cyclical, or branched;or a lipid moiety. The APF composition comprises a sialoglycopeptide, inspecific embodiments, although in alternative embodiments it comprises aglycopeptide or a peptide. In additional embodiments, the peptide hashomology to at least part of a frizzled polypeptide, such as havinghomology to at least part of a transmembrane domain of frizzled 8.

In particular embodiments, the composition is further defined ascomprising about one to about six sugar moieties; and a peptide moietyof about two to about fifteen amino acid residues, wherein one of theresidues is a linking amino acid, and wherein the peptide is linked toat least one of the sugar moieties at a heteroatom of the linking aminoacid, which may be polar, such as a serine, a threonine, a cysteine, alysine, an arginine, or a tyrosine. Thus, it is contemplated that theheteroatom linked to the sugar moiety is an oxygen, nitrogen and/or asulfur atom. In further specific embodiments, the composition comprisesthree sugar residues and nine amino acids, wherein the linking aminoacid is a serine or a threonine. In yet other specific embodiments, thecomposition comprises two sugar residues and nine amino acids

The sugar moiety comprises a naturally occurring sugar, a syntheticsugar, a derivative of a naturally occurring sugar, or a derivative of asynthetic sugar. More specifically, at least one sugar moiety is anamino sugar such as a sialic acid (N-acetylneuraminic acid) molecule,and in some embodiments, and in some embodiments, the amino sugar islinked to at least another (second) sugar via a (2,3) linkage, a (2,6)linkage, a (2,8) linkage, or a (2,9) linkage. The linkage between atleast one sugar moiety and a peptide moiety is a covalent linkage; thelinkage between a sugar moiety and a lipid moiety is a covalent linkage;and other linkages described herein may be covalent.

In specific embodiments involving more than one sugar moiety, thelinkage between one sugar moiety and another sugar moiety is a 1→3linkage, a 1→4 linkage, or a 1→6 linkage. In other embodiments, thelinkage between at least one sugar moiety and a hydrophobic moiety, suchas a peptide or a lipid, is in the alpha or beta configuration.

The peptide moiety of the composition is further defined as comprising anaturally occurring amino acid, an unnatural amino acid, a derivative ofa naturally occurring amino acid, a derivative of an unnatural aminoacid, a modified amino acid, a backbone-modifying amino acid, or amixture thereof. The peptide moiety is further defined as comprising oneor more backbone-modifying amino acids that comprise reduced peptidebonds. Modified amino acids may be further defined as a methylated aminoacid, an acetylated amino acid, a beta amino acid, or an amino acidmimetic.

The peptide moiety is about nine amino acids in length, in some aspectsof the invention, and may comprise SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, or SEQ ID NO:5. The lipid moiety in specific embodiments comprisesa saturated fatty acid such as, for example, caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid, or stearic acid; anunsaturated fatty acid such as, for example, palmitoleic acid, oleicacid, linoleic acid, ricinoleic acid, arachindonic acid; esterifiedfatty acids such as, for example, steroid alcohols in which an ester isadded to an alcohol on the lipid; omega fatty acids, including myristicand palmitic acids; or phophate-containing fats such as, for example,phosphatidylcholine and phosphatidylethanolamine; or surfactantscharacterized by having a polar head and a non-polar tail.

An APF composition of the present invention may be further defined ascomprising a label, such as a fluorescent moiety, a calorimetric moiety,or a radioactive moiety. In certain embodiments, the label is attachedto at least one of the one or more sugar moieties, such as sialic acid,glucose, galactose, N-acetylgalactosamine or N-acetylglucosamine.Alternatively, the label is attached any suitable atom in a peptide,such as within at least one of the amino acids making up the peptidemoiety, such as at a heteroatom in a serine, threonine, or cysteineamino acid subunit, or attached to the carboxyl group at the carboxylend of the peptide. Alternatively, the label is attached to at least oneof the atoms of the lipid moiety such as at a double bond in anunsaturated fatty acid (i.e., oleic acid and the like),or at a polarhead group of a lipid (alcohol).

In specific embodiments of the present invention, the APF composition isfurther defined as:

(a) Sialicacid-galactose-Nacetylgalactosamine-threonine-valine-proline-alanine-alanine-valine-valine-valine-alanine;

(b) Sialicacid-galactose-Nacetylglucosamine-threonine-valine-proline-alanine-alanine-valine-valine-valine-alanine;or

(c) Sialicacid-galactose-Nacetylglucosamine-serine-leucine-proline-alanine-alanine-valine-valine-valine-alanine.

As described further in the Examples herein, the APF compositions of thepresent invention are alternatively defined as any one of (a), (b) or(c) without a sialic acid molecule.

The composition of (a) may be further defined as having one (or one ormore) of the following: the sialic acid is linked to galactose via a 2,3linkage; the galactose is linked to the N-acetylgalactosamine via a 1,3linkage; and/or the N-acetylgalactosamine is linked to threonine via an0 linkage in an alpha configuration.

The composition of (b) may be further defined as having one (or one ormore) of the following: the sialic acid is linked to galactose via a 2,3linkage; the galactose is linked to the N-acetylglucosamine via a 1,4linkage; and/or the N-acetylglucosamine is linked to threonine via an 0linkage in an alpha configuration.

The composition of (c) may be further defined as having one (or one ormore) of the following: the sialic acid is linked to galactose via a 2,3linkage; the galactose is linked to the N-acetylglucosamine via a 1,4linkage; and/or the N-acetylglucosamine is linked to serine via an 0linkage in an alpha configuration.

In some embodiments of the present invention, the composition furthercomprises a delivery vehicle, such as a liposome, a cell, a conjugatedmolecule, a nanoparticle, or a mixture thereof. The conjugated moleculemay comprise an antibody, a peptide, folate, polyethylene glycol, adrug, a liposome, a microsphere, or lactosaminated human albumin. Thenanoparticle may comprise colloidal gold.

The composition may be comprised in a pharmaceutically acceptableexcipient. The composition may also reversibly arrest cellproliferation. In specific embodiments, the composition is furtherdefined as comprising activity for arresting cell cycling primarily inG2 or M phase or both.

In other embodiments of the present invention, there is an isolatedpeptide selected from the group consisting of SEQ ID NO:1; SEQ ID NO:3;SEQ ID NO:4; SEQ ID NO:5, or a functional derivative thereof. Thefunctional derivative thereof may be further defined as comprising aconservative substitution at one or more amino acids of the peptide. Theconservative substitution may be further defined as a substitution atserine, threonine, proline, or a combination thereof. In specificembodiments, the conservative substitution comprises a hydrophobicconservative substitution, such as a substitution at one or morealanines, one or more valines, one or more prolines; or a combinationthereof. In specific embodiments, a function of peptides of theinvention contemplated here is for use as a standard in a kit (such asto quantify APF in samples; as an antiproliferative factor; and/or as ameans for inducing antibody production.

In additional embodiments of the present invention, there is an isolatedpolynucleotide encoding SEQ ID NO:1, which may be further defined as SEQID NO:2.

The polynucleotides may be further defined as having one or more of thefollowing: a codon for threonine selected from the group consisting ofACA, ACC, ACG, and ACU; a codon for one or more valines selected fromthe group consisting of GUA, GUC, GUG, and GUU; a codon for prolineselected from the group consisting of CCA, CCC, CCG, and CCU; and acodon for one or more alanines selected from the group consisting ofGCA, GCC, GCG, and GCU.

In another embodiment, there is an isolated polynucleotide encoding SEQID NO:3, such as one further defined as having one or more of thefollowing: a codon for serine selected from the group consisting of AGC,AGU, UCA, UCC, UCG, and UCU; a codon for one or more valines selectedfrom the group consisting of GUA, GUC, GUG, or GUU; a codon for prolineselected from the group consisting of CCA, CCC, CCG, or CCU; and a codonfor one or more alanines selected from the group consisting of GCA, GCC,GCG, or GCU.

In an additional embodiment, there is an isolated polynucleotideencoding SEQ ID NO:4, such as one further defined as having one or moreof the following: a codon for threonine selected from the groupconsisting of ACA, ACC, ACG, and ACU; a codon for one or more valinesselected from the group consisting of GUA, GUC, GUG, and GUU; a codonfor proline selected from the group consisting of CCA, CCC, CCG, andCCU; a codon for one or more alanines selected from the group consistingof GCA, GCC, GCG, and GCU; and a codon for leucine selected from thegroup consisting of UUA, UUG, CUA, CUC, CUG, and CUU.

In another embodiment of the present invention, there is an isolatedpolynucleotide encoding SEQ ID NO:5, such as one further defined ashaving one or more of the following: a codon for serine selected fromthe group consisting of AGC, AGU, UCA, UCC, UCG, and UCU; a codon forleucine selected from the group consisting of UUA, UUG, CUA, CUC, CUG,and CUU; a codon for proline selected from the group consisting of CCA,CCC, CCG, and CCU; a codon for one or more alanines selected from thegroup consisting of GCA, GCC, GCG, and GCU; and a codon for one or morevalines selected from the group consisting of GUA, GUC, GUG, and GUU.

In additional embodiments of the present invention, there is a kit,comprising the APF composition housed in a suitable container. There maybe a kit for use in diagnosing a bladder disorder in a subject,comprising an antibody composition that binds immunologically to anepitope of a peptide, sugar, or glycopeptide epitope of anantiproliferative factor, said antibody composition housed in a suitablecontainer. In specific embodiments, the antiproliferative factor is aurinary bladder antiproliferative factor. The peptide may comprise oneor more hydrophobic amino acids.

In a specific embodiment, the antibody binds immunologically to anepitope of SEQ ID NO:1 or a glycopeptide derivative thereof and/or thepeptide comprises an epitope of SEQ ID NO:1 and is about 2 to 15 aminoacids in length; the antibody binds immunologically to an epitope of SEQID NO:3 or a glycopeptide derivative thereof and/or the peptidecomprises an epitope of SEQ ID NO:3 and is about 2 to 15 amino acids inlength; the antibody binds immunologically to an epitope of SEQ ID NO:4or a glycopeptide derivative thereof and/or the peptide comprises anepitope of SEQ ID NO:4 and is about 2 to 15 amino acids in length; theantibody binds immunologically to an epitope of SEQ ID NO:5 or aglycopeptide derivative thereof and/or the peptide comprises an epitopeof SEQ ID NO:5 and is about 2 to 15 amino acids in length; and/or theantibody binds immunologically to an epitope of SEQ ID NO:6 or aglycopeptide derivative thereof and/or the peptide comprises an epitopeof SEQ ID NO:6 and is about 2 to 15 amino acids in length.

Antibodies in the kit may include monoclonal antibodies; polyclonalantibodies, or both. The antibody may be labeled.

A kit of the present invention comprises a detection means for detectingAPF compositions in a sample and includes antibodies, markers such asfluorescent reporter molecules, and/or capture molecules that target asugar or peptide moiety of the composition, in additional embodiments.

In another embodiment of the present invention, there is a compositioncomprising antibodies or binding portions thereof, wherein saidantibodies or binding portions bind to a peptide, sugar, or glycopeptidemoiety of a urinary bladder antiproliferative factor, wherein saidantiproliferative factor comprises one or more sugar moieties. Inspecific embodiments, the antibodies or binding portions bind to apeptide moiety of the antiproliferative factor and inhibit activity ofthe APF molecule thereby. The antibodies may be polyclonal antibodies,monoclonal antibodies, or a mixture thereof. In specific embodiments,the peptide is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In an additional embodiment of the present invention, there is a methodof detecting a bladder disorder in an individual, comprising the step ofassaying for an APF composition. In specific embodiments, the bladderdisorder is interstitial cystitis. In specific embodiments, the assayingstep is non-invasive or invasive to the individual. In specificembodiments, the method is further defined as obtaining a sample fromthe individual; and assaying said sample for the presence of the APFcomposition. The sample may comprise urine, plasma, serum, tissue, or amixture thereof from the individual. The assaying step may comprisedetection of one or more sugar moieties of an APF composition. Theassaying step may comprise detection of the hydrophobic moiety of theAPF composition. The hydrophobic moiety of the composition may comprisea peptide moiety and the assaying step is further defined as comprisingintroduction to the sample of an antibody directed to an epitope of apeptide, sugar, or glycopeptide moiety of an APF composition. Inspecific embodiments, the assaying method comprises ELISA, Western blot,immunoblot, radioimmunoassay, immunohistochemistry, or otherantibody-based detection methods, including slot blots, dot blots and soforth. In other embodiments, the assaying method comprises in situhybridization or other means for detecting messenger mRNA for APF, forexample. In still further embodiments, the assaying method comprisesmass spectrometry, nuclear magnetic resonance, or othernon-immunological methods for detecting peptide species.

In an additional embodiment of the present invention, there is a methodof treating cancer, comprising the step of administering atherapeutically effective amount of an APF composition. In specificembodiments, the cancer comprises an epithelial cancer, such as bladdercancer or prostate cancer. In an additional specific embodiment, themethod further comprises an additional cancer therapy, such as surgery,chemotherapy, radiation, gene therapy, immunotherapy, or a combinationthereof.

In another embodiment of the present invention, there is a method oftreating a bladder disorder, comprising the step of administering atherapeutically effective amount of an APF composition. In a specificembodiment, the method further comprises an additional bladder disordertherapy. In a specific embodiment, the bladder disorder comprisesbladder cancer. In an additional specific embodiment, the method furthercomprises an additional cancer therapy, such as surgery, chemotherapy,radiation, gene therapy, immunotherapy, or a combination thereof.

In an additional embodiment, there is a method of treating ahyperplasia, comprising the step of administering a therapeuticallyeffective amount of an APF composition. In a specific embodiment, themethod further comprises an additional therapy for the hyperplasia, suchas an epithelial hyperplasia or a fibroblast hyperplasia.

In another embodiment, there is a method of enhancing cancer treatmentof an individual, comprising administering to the individual atherapeutically effective amount of an APF composition. Administrationof the composition may enhance chemotherapy, radiotherapy,immunotherapy, gene therapy, or a combination thereof. The compositionmay be administered prior to the cancer treatment being enhanced,concomitant with the cancer treatment being enhanced, subsequent to thecancer treatment being enhanced, or a combination thereof.

In an additional embodiment, there is a method of inhibitingangiogenesis in an individual, comprising administering to theindividual a therapeutically effective amount of an APF composition.

In another embodiment, there is a method of inhibiting fungal growth inan individual, comprising administering to the individual atherapeutically effective amount of an APF composition.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.:

FIGS. 1A-1C show ion trap mass spectrometric analysis of APF. In FIG.1A, there is molecular mass analysis of the active peak frommicrocapillary fractionation of HPLC-purified APF. FIGS. 1B and 1Cprovide analysis following successive fragmentation of predominantspecies by collision-induced dissociation.

FIG. 2 demonstrates antiproliferative activity of APF peptide andglycosylated derivatives. Inhibition of primary normal bladderepithelial cell ³H-thymidine incorporation by synthetic APF and itsderivatives. Equimolar quantities of the peptide backbone alone (-□-),N-acetyllactosamine-α-O-Thr derivative (closed-▾-), sialylatedN-acetyllactosamine-α-O-Thr derivative (-Δ-),N-acetyllactosamine-β-O-Thr derivative (closed-♦-), andGalβ1-3GalNAc-α-O-Thr derivative (closed -●-), were applied to normalbladder epithelial cells, and ³H-thymidine incorporation was determinedand compared to incorporation in cells grown in medium containing bufferalone. All specimens were assayed in triplicate in 2-3 repeatedexperiments; data are expressed as the mean inhibition of incorporation,and vertical lines indicate standard error of the mean.

FIGS. 3A and 3B provide microcapillary reversed-phase liquidchromatography of native APF, and synthetic GlcNAc (also referred to asGalβ1-4GlcNAcα-O-TVPAAVVVA) and GalNAc (also referred to asGalβ1-3GalNAcα-O-TVPAAVVVA, in some embodiments) derivatives.Neuraminidase-treated APF was injected along with either syntheticGalGlcNAc-containing APF or synthetic GalGalNAc-containing APF and theirrelative mobilities on C18 determined.

FIG. 4 illustrates one embodiment of the structure of APF,Galβ1-3GalNAcα-O-TVPAAVVVA.

FIG. 5 shows inhibition of T24 cell proliferation by native andsynthetic APF. Native APF (estimated concentration 3-5 μg/ml based onabsorbance at 260 nm), an equivalent volume of Mock APF, syntheticGalGalNAc APF (3.0 μg/ml of glycopeptide), or an equimolar amount ofpeptide backbone alone were compared for their ability to inhibit cellproliferation by determining live cell count after 48 hours using trypanblue exclusion. All specimens were assayed in triplicate; data areexpressed as the mean decrease in cell count, and vertical linesindicate standard error of the mean.

FIG. 6 provides Northern blot analysis of mRNA encoding the APFnonapeptide. Total RNA was extracted from explanted bladder epithelialcells from 6 patients with interstitial cystitis (lanes 1, 3, 5, 7, 9,and 11) and their age-, race-, and gender matched asymptomatic controls(lanes 2, 4, 6, 8, 10, and 12). The membrane was then incubated withDIG-labeled probes for APF mRNA and beta actin mRNA and developed usinga chemiluminescent substrate.

FIG. 7 demonstrates dose-dependent binding to HPLC-purified native APFof purified rabbit antibodies raised against synthetic APF.

FIG. 8 demonstrates antiproliferative activity against normal humanbladder epithelial cells of 2 different peptides (#1, corresponds to SEQID NO:1, and #4, corresponds to SEQ ID NO:5) and their sugarderivatives, having two (#2 and #5, each unsialyated) and three (#3 and#6) sugar moieties.

FIG. 9 shows APF inhibitory activity against LNCaP prostate cancer cellsin vitro.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The term “alpha configuration” α as used herein refers to structuralrelationships in carbohydrate chemistry, wherein the anomeric group isin the axial configuration when the conformational formulation of thepyranose ring is used. Conversely, the term “beta configuration” βrefers to that arrangement in which the anomeric group is equatorial.

The term “antiproliferative factor” as used herein refers to a moleculecomprised of one or more sugar moieties and/or a hydrophobic moiety,wherein the molecule is characterized by the ability to inhibit cellproliferation. In specific embodiments, the inhibiting activitycomprises inhibiting epithelial cell proliferation, such as bladderepithelial cell proliferation. In further specific embodiments, thehydrophobic moiety is a peptide or a lipid. In specific embodiments, thehydrophobic nature facilitates nonspecific association with a membrane,or specific or nonspecific interaction with a hydrophobic pocket of amembrane or cytoplasmic receptor, for example. In specific embodiments,the association with a membrane comprises insertion into a membrane. Themembrane may be any kind of membrane, although in particular aspects ofthe invention it is a plasma membrane. In further specific embodiments,the peptide is hydrophobic in part and comprises enough hydrophobicityto facilitate association of APF with a membrane.

The term “backbone-modifying amino acid” is known in the art and isdiscussed as follows. Normal peptide or protein backbone is formed frompolymerization of alpha amino acids, which have the amino group on thecarbon adjacent to the carboxyl group. This produces a polymer in whichthe repeating unit is —[NHCH(R)C(O)]—, wherein R is the sidechain thatmakes each amino acid different, but the repeating unit that forms theback bone is as shown. If the amino group is moved to a differentcarbon, for example the beta-carbon of alanine, the backbone is nolonger natural but still has many of the properties of peptides.Proteolytic enzymes do not recognize altered backbones. Beta-alanine orgamma-butyric acid are common backbone altering amino acids. They arenot naturally found in peptides or proteins.

The term “bladder disorder” as used herein refers to an abnormalcondition of the urinary bladder.

The term “conservative substitution” as used herein refers to replacingan amino acid in a peptide or polypeptide with a different amino acid ofa similar chemical nature. For example, a nonpolar amino acid may beconservatively substituted with another nonpolar amino acid. In specificembodiments, a hydrophobic amino acid may be substituted with anotherhydrophobic amino acid.

The term “epithelial cancer” as used herein refers to a cancer in atissue originating from epithelial cells of the tissue. For example,epithelial cancer may comprise urinary bladder; ureter; lung; heart;gastrointestinal tract (including the stomach, small intestine, largeintestine, rectum, liver, pancreas and gall bladder); spleen; malereproductive tract, including the seminal vesicles, prostate,bulbourethral gland, vas deferens, epididymis, testes, and penis; femalereproductive tract, including the ovaries, Fallopian tubes, uterus,cervix, and vagina; kidneys; adrenal glands; thymus; thyroid; skin; bone(including synovium); ocular tissues (including cornea, retina, andlens); cochlea; breast tissue; lymph nodes; oral mucosa (includinggingival), salivary gland, parotid gland; and nasopharygeal mucosa(including sinus mucosa), for example.

The term “heteroatom” as used herein refers to an atom in an organicmolecule that is other than carbon or hydrogen.

The term “hydrophobic” as used herein refers to lacking affinity forwater.

The term “hydrophobic amino acid” as used herein refers to amino acidsthat are unable to form hydrogen bonds with water because they have no,or very small, electrical charges in their structure. In aqueoussolution, hydrophobic amino acids disrupt the hydrogen bonding structurethat is formed among water molecules, given that they are unable tocontribute to it. Hydrophobic amino acids vary in size, and the majorityof hydrophobic amino acids have a side chain that is purely hydrocarbon.Other things being equal, a larger hydrophobic side chain will be morestrongly hydrophobic than a smaller one. Specific examples ofhydrophobic amino acids include those that comprise aliphatichydrocarbon side chains, such as alanine, valine, leucine, orisoleucine; aromatic side chains, such as phenylalanine or tryptophan;sulfur-comprising side chains, such as methionine; and/or imino acids,such as proline, for example. In particular embodiments, hydrophobicamino acids are considered to be alanine, valine, leucine, andisoleucine.

The term “hyperplasia” as used herein refers to the abnormalproliferation of normal cells in normal arrangement in a tissue.

The term “inhibitor of an antiproliferative factor” as used hereinrefers to an inhibitor of any APF as encompassed by the invention. Inspecific embodiments, the inhibitor degrades APF, blocks its effect oncell proliferation, that it is a growth factor, or a combinationthereof, for example. In further specific embodiments, the inhibitorcomprises an antibody, small interfering RNA, oligonucleotide, smallmolecule, and so forth, for example.

The term “peptide” as used herein refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, the peptides used herein contain atleast two amino acid residues and are less than about 50 amino acids inlength. D-amino acids are represented herein by a lower-case one-letteramino acid symbol (e.g., r for D-arginine), whereas L-amino acids arerepresented by an upper case one-letter amino acid symbol (e.g., R forL-arginine). The peptides may be cyclical, linear, branched, or acombination thereof.

“Polypeptide” as used herein refers to a polymer of at least two aminoacid residues and which contains one or more peptide bonds.“Polypeptide” encompasses peptides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

The term “protein” as used herein refers to a compound that is composedof amino acids linked by peptide bonds, but in contrast to peptides, hasa well-defined conformation, such as a well-defined three-dimensionalconformation. Proteins, as opposed to peptides, generally consist ofchains of 50 or more amino acids.

The term “purified” as used herein, is intended to refer to aglycoprotein composition, wherein the glycoprotein is purified to anydegree relative to its naturally-obtainable state, i.e., in this case,relative to its purity within a eukaryotic cell or within a fluid suchas cell medium or supernatant, or a biological fluid such as urine,serum, or plasma.

A “subunit,” as used herein, is a monomeric unit that is joined to forma larger polymeric compound. The set of amino acids are an example ofsubunits. Each amino acid shares a common backbone (—C—C—N—), and thedifferent amino acids differ in their sidechains. The backbone isrepeated in a polypeptide. A subunit represents the shortest repeatingpattern of elements in a polymer backbone. For example, two amino acidsof a peptide are not considered one subunit because two amino acidswould not have the shortest repeating pattern of elements in the polymerbackbone.

The term “terminal amino acid” as used herein refers to the amino acidon the end of a linear peptide of an APF molecule, and may refer to aN-terminal amino acid or a C-terminal amino acid.

The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of an organism including but not limitedto a mammalian organism. Such agents may take the form of ions, smallorganic molecules, peptides, proteins or polypeptides, glycopeptides(and other modified peptides), oligonucleotides, and oligosaccharides,for example.

The term “therapeutically effective amount” as used herein refers to theamount of a composition utilized alone or in combination with anothercompound for a therapeutic purpose that results in ameliorating at leastone symptom or objective finding (sign) of the medical condition beingtreated. A skilled artisan recognizes that the invention is useful forproviding less than a complete cure, so long as one or more symptoms orsigns are alleviated. For example, in treating a bladder condition, atherapeutically effective amount would include the amount thatfacilitates any or all of the following: decrease (or by inhibition, anincrease) in cell proliferation; reduction in pain, urgency, orfrequency of urination; reduction in the amount, degree and/or intensityof thinning and/or ulceration of the bladder epithelial lining; and soforth.

The term “urinary bladder” as used herein refers to a distensiblemembranous sac that serves for the temporary retention of the urine ofan individual. Normally it resides in the pelvis in front of the rectum,and it receives the urine from the two ureters, discharging it atintervals into the urethra through an orifice closed by a sphincter. Theorgan is lined with transitional hypoblastic epithelium.

The term “urinary bladder antiproliferative factor” as used hereinrefers to an antiproliferative factor as described herein that isassociated primarily with the urinary bladder. It may be associated witha cell of the bladder, such as with an epithelial cell, and this thenmay be referred to as a “urinary bladder epithelial cellantiproliferative factor”. The factor may be identified within one ormore bladder epithelial cells or it may be identified followingsecretion from one or more cells, or both. In addition, or alternativeto, the factor may be suspended in urine within a bladder or in urineexcreted therefrom, or both. Such an association of the factor with theurinary bladder may permit the detection of the APF as diagnostic for abladder condition, such as interstitial cystitis, for example. Althoughin some embodiments APF is located in the urinary bladder, inalternative embodiments the APF molecule is also associated with serum,plasma, or other tissue.

II. The Present Invention

Negative growth factors are thought to be important for normal contactinhibition of eukaryotic cells, and a decrease in their activity hasbeen associated with malignant cell growth. However, abnormallyincreased production of growth inhibitors has been implicated in only afew human disease states, including bone marrow failure (aplastic anemiaand myelodysplastic syndrome), nontoxic goiter, chronic venous ulcers,ischemic manifestations associated with systemic sclerosis and delayedhealing of gastric ulcers. In specific embodiments of the presentinvention, additional diseases are also caused by inappropriateinhibition of cell proliferation required for maintaining normal tissueintegrity, which in solid tissues may be manifest as thinning or absenceof specific cell layers and disruption of normal tissue architecture.One such class of diseases may include bladder disorders, such asinterstitial cystitis.

Approximately one million people in the United States suffer frominterstitial cystitis, a chronic painful urinary bladder disordercharacterized by thinning or ulceration of the bladder epitheliallining; its etiology is unknown. As described herein, the presentinvention is directed to a novel glycosylated frizzled-related peptideinhibitor of cell proliferation that is secreted specifically by bladderepithelial cells from patients with this disorder. Thisantiproliferative factor (APF) profoundly inhibits bladder cellproliferation via regulation of cell adhesion protein and growth factorproduction. The structure of APF was deduced using ion trap massspectrometry, enzymatic digestion, lectin affinity chromatography, andtotal synthesis, and confirmed by coelution of native and synthetic APFderivatives on microcapillary LC/MS. The standard form of APF wasdetermined to be an acidic, heat stable sialoglycopeptide whose peptidechain has 100% homology to the putative 6th transmembrane domain offrizzled 8. Both synthetic and native APF had identical biologicalactivity in normal bladder epithelial cells and T24 bladder cancercells. Northern blot analysis indicated binding of a probe containingthe sequence for the frizzled 8 segment with mRNA extracted from cellsof patients with interstitial cystitis but not controls and antibodiesraised against synthetic APF peptide bound to purified native APF in adose-dependent manner. APF is therefore the first frizzled-relatedpeptide growth inhibitor shown to contain exclusively a transmembranesegment of a frizzled protein, and is a potential biomarker forinterstitial cystitis.

The reported results demonstrate that the APF made specifically bybladder epithelial cells explanted from patients with interstitialcystitis is a uniquely modified frizzled 8-related sialoglycopeptidewith a peptide structure that bears 100% homology to the sixthtransmembrane segment of this G-protein-coupled Wnt ligand receptor(Saitoh et al., 2001). In some embodiments of the present invention,this small secreted frizzled-related peptide is normally expressed inhuman bladder epithelial cells during embryogenesis, and in analternative embodiment it is an abnormal variant of a frizzled proteinthat is produced only by bladder epithelial cells from these patients.However, its specificity for this disorder as well as its identificationas a secreted frizzled-related peptide growth inhibitor indicates itsrole in the pathogenesis of this disease. Identification of APF alsoallows for the possible development of treatment for interstitialcystitis based on inhibition of APF production or activity, orstimulation of APF breakdown. In addition, exclusive expression of APFin adults by bladder epithelial cells from interstitial cystitispatients in some embodiments provides a direct noninvasive diagnostictest for this disorder.

To date, two disease states have been noted to be associated withabnormally increased expression of secreted frizzled-related proteinsother than APF: overload-induced heart failure, in which mRNA for twosuch proteins have been shown to be elevated in failing ventricles ascompared to control hearts (Schumann et al., 2000), and degenerativeretinal disease, in which a secreted frizzled-related protein and itsmRNA expression are greatly elevated (Jones et al., 2000). Thepreviously identified secreted frizzled-related proteins contain acysteine-rich extracellular domain, allowing them to inhibit Wntsignaling by one of two mechanisms: by binding to the Wnt ligand, or byforming nonsignalling dimers with frizzled receptors (Bafico et al.,1999). APF is therefore the first frizzled-related growth inhibitor thatbears homology only to a transmembrane portion of a frizzled receptor,suggesting that growth inhibition by frizzled-related proteins may alsooccur via additional mechanisms. APF is also the smallest secretedfrizzled-related peptide identified to date, with previously describedfrizzled-related peptides having molecular weights of 33.5-39.9 kDa(Jones and Jomacy, 2002).

Microarray analysis previously indicated that APF decreases theexpression of several genes including Jun N-terminal kinase in bladderepithelial cells (Keay et al., 2003), suggesting the possibility thatAPF may interfere with non-canonical Wnt signaling mediated via afrizzled receptor protein (Pandur et al., 2002). However, linkage of thecytoskeleton to its substratum includes binding of the actin-catenincomplex to E-cadherin, a protein whose expression has also been shown tobe significantly upregulated by APF in bladder epithelial cells (Keay etal., 2003), and which is known to inhibit canonical Wnt signaling inboth urothelial carcinoma and normal urothelial cells in vitro(Thievessen et al., 2003). Not to be bound to any theory, thus, inspecific embodiments APF mediates its antiproliferative effects viainhibition of canonical or non-canonical Wnt pathway(s) in eitherbladder carcinoma or normal bladder cells, for example.

Although other potent sialylated small glycopeptide growth inhibitorshave been isolated from serum or brain of normal mammals and shown toinhibit proliferation of both normal and malignant cells from a varietyof tissues (Auger et al., 1989; Moos et al., 1995), no structural dataare available for any of these natural growth inhibitors and theirrelationship to frizzled-related proteins or any specific signalingpathway(s) has not been determined. APF is therefore the first of thisclass of growth inhibitors to be completely characterized as well as thefirst to be synthesized. The requirement of the α-O-linkedN-acetylhexosamine structure for antiproliferative activity andregulation of growth factor production by APF suggests that APF may bindto a specific cellular receptor(s), as suggested for anothersialoglycopeptide growth inhibitor (Sharifi and Johnson, 1987). Theextremely hydrophobic nature of the peptide moiety of APF furthersuggests the possibility that it may interact with the plasma membraneand expose a distinct conformation of the sugar moiety at the cellsurface.

III. Antiproliferative Factor (APF)

The present invention encompasses compositions and methods associatedwith antiproliferative factor (APF). APF comprises a glycopeptide thatinhibits proliferation of bladder epithelial cells, skin fibroblasts,and other epithelial cells including prostate cells, and in someembodiments is generated by bladder epithelial cells, such as thoseassociated with interstitial cystitis. In particular embodiments, thecompound is present in the urine of individuals having interstitialcystitis. In other embodiments, the compound is generated orbiosynthesized by tissues and cells other than urinary bladder tissueand cells. In one aspect of the invention, the compound is considered atoxin, a negative growth factor, or both.

APF was identified because of its ability to inhibit the growth of cellsthat line the bladder wall, in specific embodiments by altering theproduction of several proteins by these cells, such as specific growthfactors and cell adhesion proteins. Not to be bound to any theory, infurther embodiments, APF causes interstitial cystitis in which thebladder lining is generally thin and/or ulcerated.

Thus, as used herein the term “APF” refers to a class of compoundswherein the structure in FIG. 4 is merely the prototypical APF and otherrelated compositions are encompassed, so long as they are diagnostic fora bladder condition and/or are useful as a direct or indirect target totreat a disorder associated with abnormally increased cellproliferation, and their inhibitors are useful for treating disordersassociated with decreased cell proliferation.

Although in particular aspects of the invention APF comprises thestructure provided in FIG. 4, this is merely one embodiment of theinvention. A skilled artisan recognizes that the structure in FIG. 4 maybe secreted by bladder epithelial cells and is therefore diagnostic of abladder disorder such as interstitial cystitis. However, in someembodiments a similar but non-identical structure of APF is diagnosticof a bladder disorder. APF compositions herein encompass both isolatednatural APF, synthetic versions thereof, derivatives thereof, or amixture thereof. Furthermore, the compounds of the present invention maybe made using synthetic means or isolated from a natural source, such asbladder epithelial cells, their extracellular medium, tissue or bodilyfluids such as urine, serum, or plasma.

Furthermore, inhibition of a molecule having the structure of APF inFIG. 4 is useful for the invention, but inhibition of a molecule havinga similar structure of APF may also be useful. The inhibition may bedirected to inhibiting the function of APF, such as with an antibody,for example; or for inhibiting the production of APF, such as ananti-sense oligonucleotide or small interfering RNA, for example; or forstimulating the breakdown of APF; or a combination thereof.

Thus, in specific embodiments, compositions related to the presentinvention comprise about one to about six sugar residues; and a peptideof about two to about fifteen amino acid residues, wherein thepeptide-linked to one of the sugar moieties at a linking amino acid,wherein the linking amino acid comprises a heteroatom which serves asthe linking portion of the linking amino acid. More specifically, thelinking amino acid comprises a serine, a threonine, or a cysteine. Inother specific embodiments, the compositions of the present inventioncomprises two or three sugar residues and nine amino acids and thelinking amino acid is a threonine or serine.

In one particular aspect of the invention, an APF composition maycomprise in part a hydrophobic moiety, such as a peptide, for exampleone including SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5, ora lipid. The peptide may comprise at least part of a transmembranedomain, and in particular embodiments it comprises part of frizzled 8,such as a transmembrane domain of frizzled 8. In specific embodiments,the peptide is hydrophobic.

The glycoprotein comprising a galactose covalently linked to anN-acetylglucosamine or an N-acetylgalactosamine covalently linked to apeptide of SEQ ID NO:1 or variants thereof is provided herein. The term“variants thereof” includes peptidomimetics of various types (Ahn etal., 2002). The peptides may comprise any suitable amino acids, such asL-amino acids, D-amino acids, N-methylated amino acids, or a combinationthereof, as well as peptidomimetic compounds such as unnatural aminoacids or other “peptide-like” organic constructs that mimic the specificstructural elements of a linear, cyclic, or branched peptide thatcorrespond to active peptides. The sugar moieties may be natural,synthetic, carbohydratemimetic, or a mixture thereof may be used in acomposition. Glycopeptidomimetic compounds where the sugars arecarbohydratemimetic moieties or the peptide components arepeptidomimetic moieties, or a combination of the two, are encompassed inthe invention. In specific embodiments, the sugars of the presentinvention include amino sugars.

In a particular aspect of the invention, the APF has a molecular mass of1482.8 and comprises nine amino acids and three sugar moieties in thefollowing order: (a) Sialicacid-galactose-N-acetylgalactosamine-threonine-valine-proline-alanine-alanine-valine-valine-valine-alanine;or (b) Sialicacid-galactose-Nacetylglucosamine-threonine-valine-proline-alanine-alanine-valine-valine-valine-alanine;or (c) Sialicacid-galactose-N-acetylglucosamine-serine-leucine-proline-alanine-alanine-valine-valine-valine-alanine.The composition may be further defined as having one or more of thefollowing: the sialic acid in (a) is linked to galactose via a 2,3linkage; the sialic acid in (b) is linked to galactose via a 2,3linkage; the sialic acid in (c) is linked to galactose via a 2,3linkage; the galactose in (a) is linked to the N-acetylgalactosamine viaa 1,3 linkage; the galactose in (b) is linked to the N-acetylglucosaminevia a 1,4 linkage; the galactose in (c) is linked to theN-acetylglucosamine via a 1,4 linkage; the N-acetylglucosamine is linkedto serine via an O linkage in an alpha configuration; or theN-acetylgalactosamine is linked to threonine or serine via an O linkagein an alpha configuration.

It is contemplated that the compounds of the present invention may bemodified so as to improve certain characteristics, such as solubility byadding a water soluble unit. The term “water soluble unit” means anyfunctional group imparting water solubility, including, but not limitedto, SO₃—, PO₃ ²⁻, CH₂ COO⁻, a quaternary ammonium group attached via anester or alkyl linkage such as C=O(CH₂)_(x) NAlk₃ or (CH₂)_(x) NAlk₃where Alk₃ represents three alkyl groups that are independently C1-C4alkyl and x is 1-4, (CH₂ CH₂ O)_(n) CH₂ CH₂ OX (n=1-3) wherein X may beH or CH₃, i.e., PEG or MeO-PEG. The counterion for water soluble unitsbearing a charge include, but are not limited to, metals such as alkaliand alkaline earth metals, and halogens.

Certain compounds of the present invention comprise a threonine, aserine, or a cysteine at the N- terminus or any functional equivalent.Non-limiting examples of functional equivalents include a syntheticderivative having a primary or secondary or tertiary alcohol, an ester,a carboxylic acid, an ether, a thiol, a thiolate, or any functionalgroup enabling for covalent linkage with a sugar molecule, provided themolecule retains biological function.

Other functionalities contemplated in “derivatives” of the presentinvention include isomers of any of the sugars or amino acids, whetherpositional, structural, or stereoisomers. Other substituents known tothose skilled in the chemical arts may be provided, so long as thebiological function of the molecule is retained.

IV. Diagnostic Embodiments

In specific embodiments of the present invention, APF compounds of thepresent invention are utilized to diagnose one or more bladderdisorders.

In specific aspects of the invention, identification of an APF compoundfor an individual indicates an increased risk for developing aparticular bladder disorder, and in some cases the symptoms of thedisease may not yet be apparent. Thus, in some embodiments, theinvention encompasses identifying a predisposition to developing abladder condition, such as interstitial cystitis, or it encompassesidentifying the presence of the disease.

In specific embodiments of the invention, the diagnosis of a bladderdisorder comprises detection of APF glycopeptide or its activity, andthe detection may be direct detection of the APF molecule or indirectdetection, such as of an intermediate, a by-product, a breakdownproduct, a derivative, and so forth. The detection of APF may employ anysuitable means in the art, although in specific embodiments thedetection utilizes an antibody to the peptide, sugar, or glycopeptidemoiety of APF; techniques such as mass spectrometry, nuclear magneticresonance, or proteomic methods to detect the peptide, sugar, orglycopeptide moiety of APF; or assays for inhibition of normal ormalignant cell metabolism or proliferation to detect APF activity, forexample. In general, assays used to detect the peptide moiety of APF ina sample derived from an individual are well-known to those of skill inthe art and include western blot analysis, ELISA assays, “sandwich”assays, radioimmunoassay, immunohistochemistry or other antibody-basedassays (see, e.g., Coligan et al., Current Protocols in Immunology 1(2),Chapter 6, (1991)). Generally, Western blotting is a technique forblotting proteins or peptides onto nictrocellulose, nylon or othertransfer membrane after they have been resolved by gel electrophoresis.The proteins can be detected by one of several methods, includingautoradiography (if labeled), or through binding to labeled,¹²⁵I-labeled or enzyme-linked antibodies, lectin or other specificbinding agents, for example.

An ELISA assay initially comprises preparing an antibody specific to theantigen, wherein the antigen comprises the peptide moiety, the sugarmoiety, or the glycopeptide of APF, and this is preferably a monoclonalantibody. Next, a reporter antibody typically is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as a radioactive moiety, fluorescent moiety or enzyme, suchas horseradish peroxidase or alkaline phosphatase. A sample is removedfrom the host and incubated on a solid support, e.g., a polystyrenedish, binding the proteins in the sample. Any free protein binding siteson the dish are then blocked by incubating with a non-specific protein,such as bovine serum albumen or milk proteins. Next, the antibody isincubated in the dish during which time the antibodies attach to anypeptide moieties, the sugar moieties, or the glycopeptide moieties ofAPF from the sample attached to the polystyrene dish. All unboundantibody is washed out with buffer. The reporter antibody is then placedin the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to the peptide moiety of APF. Unattachedreporter antibody is then washed out, and the peptide moiety, the sugarmoiety, or the glycopeptide of APF is then detected. Alternatively, theanti-APF antibody may be directly labeled, as above. In specificembodiments, the amount of APF present in a given volume of a patientsample is proportional to the detection of the peptide moiety, the sugarmoiety, or the glycopeptide thereof and is compared against a standardcurve.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assay,the peptide moiety, the sugar moiety, or the glycopeptide of APF ispassed over a solid support and allowed to bind to antibody attached tothe solid support. A second antibody is then allowed to bind to thepeptide moiety, the sugar moiety, or the glycopeptide of APF. A thirdantibody specific to the second antibody is then passed over the solidsupport and binds to the second antibody, which is thereby detected.Alternatively, the second antibody may be directly labeled, as above.

V. Therapeutic Embodiments

A skilled artisan recognizes that the APF compositions of the presentinvention may be addressed in a variety of ways to provide therapy for abladder condition. For example, in therapeutic embodiments whereinproliferation is desirable, such as with interstitial cystitis, aninhibitor of APF activity or production, or a stimulator of APFbreakdown, may be employed for therapy. In therapeutic embodimentswherein proliferation is undesirable, an APF composition may be employedfor therapy.

In one aspect of the invention, a deleterious bladder condition isassociated either indirectly or directly with reduced cellularproliferation, such as inhibited epithelial cellular proliferation. Sucha condition may result in harmful alterations to the bladder epitheliumsuch that it would be beneficial to reduce or substantially remove theinhibition of cellular proliferation. In these aspects, it is desirableto deliver to the individual with the bladder condition, such as deliversystemically to the individual or directly to bladder epithelium, aninhibitor of an APF composition. Specific examples of APF inhibitorsinclude antibodies to the peptide moieties of the APF composition, aswell as oligonucleotides, small interfering RNAs, small compounds thatblock the interaction of APF with its target, and compounds that causeor stimulate the breakdown of APF, and/or a mixture thereof. Thecomposition may be delivered by any suitable means, although in specificembodiments it is delivered via catheter, orally, intravenously,topically, subcutaneously, transcutaneously, intramuscularly,intra-jointly, parenterally, peritoneally, intranasally, intravesicallyor by inhalation. In other specific embodiments, the composition iscomprised in a pharmaceutically acceptable excipient, such as an aqueousor non-aqueous liquid. In particular aspects of the invention, it isadministered in a non-aqueous excipient due to the hydrophobic nature ofthe peptide moiety. It may be delivered alone or in a carrier, such as aliposome, encapsulated cell, viral vector, nanoparticles, biodegradablegel or polymer, implanted osmotic pump, or other suitable devices.

In another aspect of the invention, a deleterious bladder condition isassociated either indirectly or directly with increased cellularproliferation, such as increased epithelial cellular proliferation. Sucha condition may result in malignancy of the bladder epithelium, suchthat it would be beneficial to reduce in part or substantially in fullthe amount of cellular proliferation. In these aspects, it is desirableto deliver to the individual with the bladder condition, such as deliveran APF composition systemically to the individual or directly to bladderepithelium, an APF composition. The composition may be delivered by anysuitable means, although in specific embodiments it is delivered viacatheter, orally, intravenously, topically, subcutaneously,transcutaneously, intramuscularly, orally, intra-jointly, parenterally,peritoneally, intranasally, intravesically or by inhalation. In otherspecific embodiments, the composition is comprised in a pharmaceuticallyacceptable excipient. It may be delivered alone or in a carrier, such asa liposome, encapsulated cell, viral vector, nanoparticles,biodegradable gel or polymer, implanted osmotic pump, or other suitabledevices.

In a particular embodiment, an APF composition of the present inventionmay be administered to an individual with any kind of cancer, includingepithelial cancers. In specific embodiments, there is a malignancy ofthe bladder epithelium, which may be referred to herein as bladdercancer. In specific embodiments, there is a cancer therapy additional tothe APF treatment, such as gene therapy, chemotherapy, radiation,surgery, immunotherapy, or a combination thereof.

VI. Bladder Disorders

Although the present invention may be useful for any medical conditionfor which APF provides therapy, in specific embodiments the presentinvention is useful for one or more bladder disorders. Although the term“bladder disorder” refers to any abnormal condition of the urinarybladder, in specific embodiments the bladder disorder comprisesinterstitial cystitis, bladder cancer, either as a primary or secondarycancer, chronic pelvic pain syndrome, irritable bladder syndrome,urethral syndrome, painful bladder syndrome, chronic nonbacterialprostatitis, and other bladder conditions, for example.

In specific embodiments of the present invention, there are methods andcompositions related to interstitial cystitis. Typical symptoms ofinterstitial cystitis include pain, which can be in the abdominal,urethral or vaginal area and is also frequently associated with sexualintercourse; urgency, which includes the sensation of having to urinateimmediately and may also be accompanied by pressure and/or spasms; andincreased frequency of urination, which can be day and/or nightfrequency of urination.

Diagnosis of intersitial cystitis is heretofore performed usingcystoscopy, and hydro-distention and biopsies are normally performed atthe same time. Examination by cytoscopy of a typical bladder havinginterstitial cystitis may identify submucosal pinpoint hemorrhages(glomerulations), thinning of the epithelium and/or Hunner's ulcers; insome cases, inflammation may also be present. Thus, there isconsiderable pain when urine enters into the bladder of an IC patient,making it very difficult for patients with interstitial cystitis to beable to hold urine in their bladder, due to the burning, stinging andpain.

Current therapies include oral medications, such as Elmiron,Amitriptyline (Elavil) Atarax, Neurontin, Ditropan, Prozac, Cimetidine.In specific embodiments of the invention, therapeutic agents associatedwith the present invention are used either alone or in conjunction withone or more of these or similar medications. In specific embodiments,the patients also suffer with various other syndromes includingfibromyalgia, urethral syndrome, vulvodynia, irritable bowel syndrome,chronic fatigue syndrome, allergies, and other auto-immune disorders,such as scleroderma.

VII. Pharmaceutical Compositions

The present invention is also directed to pharmaceutical compositionsfor use in treating or ameliorating bladder conditions, such asinterstitial cystitis, bladder cancer, epithelial hyperplasia ormalignancies of epithelial origin, of fibroblast hyperplasia ormalignancy, other solid tumors, or lymphoreticular malignancies. Thecompounds of the present invention are also contemplated for use as anadjuvant treatment for bladder cancer or other malignancies, as anantiangiogenic agent or an antifungal agent. It is further contemplatedthat the compounds of the present invention may be used to generateantibodies, antisense oligonucleotides, small interfering RNA, or agentsthat block the interaction of APF with its target for the treatment ofinterstitial cystitis or other disorders related to cell proliferation.

Such methods generally involve administering a pharmaceuticalcomposition comprising an effective amount of the glycopeptides of thepresent invention. Other exemplary compositions include an effectiveamount of an oligonucleotide encoding the nonapeptide of a sequence asessentially set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or a small interfering RNA comprising part or all of a sequenceencoding peptides as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,or SEQ ID NO:5, for example.

Where the invention is directed to treating with the compounds of thepresent invention, administration of the compounds of the invention witha suitable pharmaceutical excipient as necessary can be carried out viaany of the accepted modes of administration. The compounds may becomprised in a pharmaceutically acceptable excipient, which may beconsidered as a molecular entity and/or composition that does notproduce an adverse, allergic and/or other untoward reaction whenadministered to an animal, as appropriate. It includes any and/or allsolvents, dispersion media, coatings, antibacterial and/or antifungalagents, isotonic and/or absorption delaying agents and/or the like. Theuse of such media and/or agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media and/oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated.

Thus, administration can be, for example, intravenous, topical,subcutaneous, transcutaneous, intramuscular, oral, intra-joint,parenteral, peritoneal, intranasal, intravesical or by inhalation.Suitable sites of administration thus include, but are not limited to,skin, bronchial, gastrointestinal, anal, vaginal, eye, bladder, and ear.The formulations may take the form of solid, semi-solid, lyophilizedpowder, or liquid dosage forms, such as, for example, tablets, pills,capsules, powders, solutions, suspensions, emulsions, suppositories,retention enemas, creams, ointments, lotions, aerosols or the like,preferably in unit dosage forms suitable for simple administration ofprecise dosages.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, and the like. Preferably, the composition will beabout 5% to 75% by weight of a compound or compounds of the invention,with the remainder consisting of suitable pharmaceutical excipients.Appropriate excipients can be tailored to the particular composition androute of administration by methods well known in the art, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co.,Easton, Pa. (1990).

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. The composition may take the form of a solution, suspension,tablet, pill, capsule, powder, sustained-release formulation, and thelike.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet or capsule, and thus, the composition can contain, alongwith the biologically active conjugate, any of the following: a diluentsuch as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof, a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Liquid compositions can be prepared by dissolving or dispersing compound(about 0.5% to about 20%), and optional pharmaceutical adjuvants in acarrier, such as, for example, aqueous saline (e.g., 0.9% w/v sodiumchloride), aqueous dextrose, glycerol, ethanol and the like, to form asolution or suspension, e.g., for intravenous administration. The activecompounds may also be formulated into a retention enema.

If desired, the composition to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, such as, for example, sodium acetate,sorbitan monolaurate, or triethanolamine oleate.

For topical administration, the composition is administered in anysuitable format, such as a lotion or a transdermal patch. For deliveryby inhalation, the composition can be delivered as a dry powder (e.g.,Inhale Therapeutics) or in liquid form via a nebulizer.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences, supra., and similar publications. The composition to beadministered will, in any event, contain a quantity of the pro-drugand/or active compound(s) in a pharmaceutically effective amount forrelief of the condition being treated when administered in accordancewith the teachings of this invention.

Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person. However, it is possible that an effective dose ofAPF, especially if administered directly into the bladder, may beoutside of this range.

Stability of the conjugate can be further controlled by chemicalalterations, including D amino acid residues in the polypeptide chain aswell as other peptidomimetic moieties. Furthermore, stability of theconjugates could also be enhanced by unnatural carbohydrate residues.

VIII. Combination Treatments

In order to increase the effectiveness of an APF composition for thetreatment of cancer in an individual, such as a patient, it may bedesirable to combine these compositions with other agents effective inthe treatment of hyperproliferative disease, such as anti-cancer agents.An “anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or multiple factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy, for example, by combining it with other cancer therapies.In the context of the present invention, it is contemplated that APFcomposition therapy could be used similarly in conjunction withchemotherapeutic, radiotherapeutic, surgical, or immunotherapeuticintervention, in addition to other pro-apoptotic or cell cycleregulating agents.

Alternatively, the APF treatment may precede, follow, or both the otheragent treatment by intervals ranging from minutes to weeks. Inembodiments where the APF composition and the other agent are appliedseparately to a cell of the individual, one would generally ensure thata significant period of time did not expire between the time of eachdelivery, such that the APF composition and the other agent would stillbe able to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, for example, wherein the APFtreatment is “A” and the secondary agent, such as radio- orchemotherapy, is “B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/BB/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/BA/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the APF compositions of the present invention to apatient will follow general protocols for the administration ofchemotherapeutics, taking into account the toxicity, if any, of themolecule. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed hyperproliferative cell therapy.

-   -   A. Chemotherapy

A skilled artisan recognizes that in addition to the APF treatmentdescribed herein for the purpose of inhibiting cell growth, otherchemotherapeutic agents are useful in the treatment of neoplasticdisease. Examples of such chemotherapeutic agents include, for example,cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate, or any analogor derivative variant of the foregoing.

-   -   B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

-   -   C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with APF therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

-   -   D. Genes

In yet another embodiment, the secondary treatment is a secondary genetherapy in which a second therapeutic polynucleotide is administeredbefore, after, or at the same time as an APF molecule, having a combinedanti-hyperproliferative effect on target tissues. A variety of proteinsare encompassed within the invention, including inhibitors of cellularproliferation, such as tumor suppressors, including p53; and/orregulators of programmed cell death, such as Bc1-2.

-   -   E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

-   -   F. Other agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adehesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5 /TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adehesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

IX. Kits

Diagnostic and/or therapeutic kits associated with the glycoproteins ofthe present invention comprise another aspect of the present invention.Such kits will generally contain, in suitable container means, an APFmolecule of the present invention, such as a glycoprotein comprising apeptide having a sequence as, for example, but not limited to, oneessentially set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQID NO:5, or a means to detect such as molecule. The kit may have asingle container means that contains the APF composition or it may havedistinct container means for the APF composition and other reagents thatmay be included within such kits. Other kits may comprise inhibitors ofAPF in suitable container means, such as antibodies, small interferenceRNAs, and so forth. Diagnostic kits may comprise any suitable reagentsto identify an APF composition, such as antibodies, for example.

The components of the kit may be provided as liquid solution(s), or asdried powder(s). When the components are provided in a liquid solution,the liquid solution is an aqueous or non-aqueous solution, with asterile aqueous or non-aqueous solution being particularly preferred.When reagents or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

Kits may comprise reagents for detecting peptides having a sequence asessentially set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQID NO:5, such as is required for immunoassay. The immunodetectionreagent will typically comprise a label associated with the antibody orantigen, or associated with a secondary binding ligand. Exemplaryligands might include a secondary antibody directed against the firstantibody or antigen or a biotin or avidin (or streptavidin) ligandhaving an associated label. Of course, a number of exemplary labels areknown in the art and all such labels may be employed in connection withthe present invention. The kits may contain antibody-label conjugateseither in fully conjugated form, in the form of intermediates, or asseparate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which the antigenor antibody may be placed, and preferably suitably aliquoted. Where asecond binding ligand is provided, the kit will also generally contain asecond vial or other container into which this ligand or antibody may beplaced. The kits of the present invention will also typically include ameans for containing the antibody, antigen, and reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

X. APF Antibodies

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a proteinaceous or sugar composition, such as apeptide, a sugar, a glycopeptide, or a combination thereof of theinvention. The peptide may be comprised as part of a larger composition,such as an APF of the present invention.

An antibody can be a polyclonal or a monoclonal antibody. In a preferredembodiment, an antibody is a monoclonal antibody. Means for preparingand characterizing antibodies are well known in the art (See, e.g.,Howell and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a peptide, sugar or glycopeptide of the presentinvention, with or without an adjuvant or other such molecules thatenhance immune responses (e.g. KLH) and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically an animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a chicken, a hamster or aguinea pig. Because of the relatively large blood volume of rabbits, arabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for APF peptides,sugars, glycopeptides, and particularly those represented by SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, variants and epitopesthereof, may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of APF can be used to immunize one or moreexperimental animals, such as a rabbit, chicken, or mouse, which willthen proceed to produce specific antibodies against the peptide, sugar,or glycopeptide regions of APF. Polyclonal antisera may be obtained,after allowing time for antibody generation, simply by bleeding theanimal and preparing serum samples from the whole blood.

To obtain monoclonal antibodies, one would also initially immunize anexperimental animal, often preferably a mouse, with an APF composition.One would then, after a period of time sufficient to allow antibodygeneration, obtain a population of spleen or lymph cells from theanimal. The spleen or lymph cells can then be fused with cell lines,such as human or mouse myeloma strains, to produce antibody-secretinghybridomas. These hybridomas may be isolated to obtain individual cloneswhich can then be screened for production of antibody to the desired APFpeptide, sugar, or glycopeptide.

Following immunization, spleen cells are removed and fused, using astandard fusion protocol with plasmacytoma cells to produce hybridomassecreting monoclonal antibodies against APF and/or APF peptides.Hybridomas which produce monoclonal antibodies to the selected antigensare identified using standard techniques, such as ELISA and Western blotmethods. Hybridoma clones can then be cultured in liquid media and theculture supernatants purified to provide the APF-specific monoclonalantibodies.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods, as well as other procedures which mayutilize antibody specific to APF epitopes.

Additionally, it is proposed that monoclonal antibodies specific to theparticular peptide may be utilized in other useful applications. Forexample their use in immunoabsorbent protocols may be useful inpurifying native or recombinant APF species or variants thereof.

In general, both poly- and monoclonal antibodies against APF may be usedin a variety of embodiments. For example, they may be employed inantibody cloning protocols to obtain cDNAs or genes encoding APF orrelated proteins. This may also be used to select for hybridoma cells orlymphocytes that recognize the APF epitopes or make antibodies orsegments thereof that recognize APF epitopes. They may also be used ininhibition studies to analyze the effects of APF in cells or animals.Anti-APF antibodies will also be useful in immunolocalization studies toanalyze the distribution of APF peptides during various cellular events,for example, to determine the cellular or tissue-specific distributionof the APF peptide or glycopeptide under different physiologicalconditions. A particularly useful application of such antibodies is inpurifying native or recombinant APF peptides or glycopeptides, forexample, using an antibody affinity column. The operation of all suchimmunological techniques will be known to those of skill in the art inlight of the present disclosure.

While the invention has been described with reference to certainparticular embodiments thereof, those skilled in the art will appreciatethat various modifications may be made without departing from the spiritand scope of the invention. The scope of the appended claims is not tobe limited to the specific embodiments described.

The present invention also encompasses methods of generating antibodiesto an APF composition, such as to a peptide moiety, a sugar moiety,and/or a glycopeptide moiety. The peptide moities include SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, for example.These antibodies may then be utilized for detection of a bladdercondition, such as interstitial cystitis, for example; for diagnosis ofa bladder condition; or for therapeutic purpose, such as to inhibit anAPF composition.

XI. Screening for Inhibitors of Antiproliferative Factor

Inhibitors of APF activity can also be screened using direct or indirectcell proliferation inhibition assays, for example. In the direct assays,inhibition of normal or malignant cell proliferation by APF is abrogatedin a dose-dependent manner. Cell proliferation assays, includingtritiated thymidine incorporation, bromodeoxyuridine incorporation, livecell counts, or any of a number of commercially available compounds usedto detect cell proliferation could be used for this purpose. In theindirect assay, inhibition of normal or malignant cell metabolism by APFis used as an indirect indication of cell proliferation; the inhibitionof cell metabolism by APF would then be abrogated in a dose-dependentmanner by inhibitors of APF activity.

EXAMPLE 1 Exemplary Methods and Reagents

Cell Cultures

Cystoscopy was performed under general anesthesia, and 4 mm2 pieces oftransitional epithelium with submucosa bladder tissue were obtainedusing rigid cold cup biopsy forceps from six patients who had previouslyundergone cystoscopy and fulfilled the NIDDK/NIH diagnostic criteria forinterstitial cystitis, and six age- race- and gender-matched controlswho were asymptomatic for urinary tract disease, as previously described(Keay et al., 2001; Keay et al., 2000; Keay et al., 2003a; Keay et al.,2003b; Keay et al., 1996). All patients were at least 18 years old andenrolled in accordance with guidelines of the Institutional Review Boardof the University of Maryland School of Medicine.

Explanted epithelial cells were propagated from these biopsy specimensin DMEM-F12 (Media Tech, Hemdon, Va.) with 10% heat inactivated fetalbovine serum (FBS), 1% antibiotic/antimycotic solution, 1% L-glutamine,1.0 U/ml insulin (all from Sigma, St. Louis, Mo.), and 5 μg/ml hEGF (R &D Systems, Minneapolis, Minn.) at 37° C. in a 5% CO2 atmosphere, andcharacterized by binding of AE-1/AE-3 pancytokeratin antibodies (Signet,Dedham, Mass.), as previously described (Keay et al., 2001; Keay et al.,2000; Keay et al., 2003a; Keay et al., 2003b; Keay et al., 1996).

T24 bladder carcinoma cells (ATCC #HTB 4) were purchased from theAmerican Type Culture Collection (Rockville, Md.) and cultured inMcCoy's 5A medium (Gibco-Invitrogen, Carlsbad, Calif.) containing 10%FBS, 1% glutamine and 1% antibiotic/antimycotic solution (all fromSigma).

APF Purification

APF was harvested from the supernatant of explanted patient bladderepithelial cells and purified using molecular weight fractionation, ionexchange chromatography, hydrophobic interaction chromatography, andreversed phase high performance liquid chromatography (HPLC), aspreviously described (Keay et al., 2000). Mock APF was prepared usingthe supernatant of normal control bladder epithelial cells and the samepurification procedure.

Microcapillary LC-MS/MS Analysis

Microcapillary reversed-phase liquid chromatography (mRPLC) wasperformed using an Agilent 1100 capillary LC system (AgilentTechnologies, Palo Alto, Calif.) coupled online to an ion trap massspectrometer (LCQ Deca XP, ThermoFinnigan, San Jose, Calif.).Reversed-phase separations of each sample were performed using 75μmi.d.×10 cm long fused silica electrospray ionization capillary columns(Polymicro Technologies, Phoenix, Ariz.) that were slurry packed inhouse with 3 μm, 300 Å pore size C-18 stationary phase (Vydac Hesperia,Calif.). After sample injection, the column was washed for 20 min with98% solvent A (0.1% v/v formic acid in water) and gradient elution wasconducted using a linear step gradient from 2% solvent B (0. 1% v/vformic acid in acetonitrile) to 42% solvent B in 40 minutes, then from42% to 95% B in 15 min, at a constant flow rate of 0.5 μL/min.

The ion trap mass spectrometer was operated in a data dependent mode inwhich each full mass spectrometry (MS) scan was followed by three tandemMS scans where the three most abundant molecular ions were dynamicallyselected for collision-induced dissociation (CID) using a normalizedcollision energy of 38%. The temperature of the heated capillary andelectrospray voltage were 180° C. and 1.8 kV, respectively.

³H-Thymidine Incorporation

Cell proliferation was measured by ³H-thymidine incorporation intoexplanted normal human bladder epithelial cells, as previously described(Keay et al., 2001; Keay et al., 2000; Keay et al., 2003a; Keay et al.,2003b; Keay et al., 1996). Briefly, purified native or synthetic APF (orthe appropriate mock preparation) was diluted in serum-free MEM(containing only glutamine and antibiotics/antimycotics) and applied tothe cells; cell controls received serum-free MEM alone. Cells were thenincubated at 37° C. in a 5% CO₂ atmosphere for 48 hours. The cells werethen labeled with 1 μCi/well ³H-thymidine for 4 hours, trypsinized,insoluble cell contents harvested and methanol-fixed onto glass fiberfilter paper, and the amount of radioactivity incorporated determined.Significant inhibition of 3H-thymidine incorporation was defined as amean decrease in counts per minute of greater than 2 standard deviationsfrom the mean of control cells for each plate.

Cell Count Determination

For cell count experiments, explanted normal human bladder epithelialcells, LNCaP prostate carcinoma cells, or T24 bladder carcinoma cellswere plated onto Corning 24 well tissue culture plates (VWR ScientificProducts) in MEM (primary bladder cells), DMEM (LNCaP cells), or McCoy's5A medium (T24 cells) containing 10% FBS, 1% antibiotic/antimycoticsolution, 1% L-glutamine at a density of 1×10⁴ cells/well and incubatedat 37° C. in a 5% CO₂ atmosphere overnight. On the next day the mediumwas removed and replaced with serum-free MEM, DMEM, or McCoy's mediumcontaining 1% antibiotic/antimycotic solution and 1% L-glutamine (allthree cell types). HPLC-purified or synthetic APF (or their mockpreparations) were then added to the medium and cells further incubatedat 37° C. with 5% CO₂. Forty-eight hours later the culture supernatantwas removed for growth factor analysis by ELISA, cells were trypsinized,stained with Trypan blue (Sigma) and counted using a hemacytometer.

ELISAs

HB-EGF-ELISAs were performed as previously described (Keay et al., 2001;Keay et al., 1998), coating 96 well Immulon II plates (DynatechLaboratories, Chantilly, Va.) with 200 λ culture supernatant at 40° C.overnight. Plates were subsequently rinsed, blocked, and anti-HB-EGFantibody (1 μg/ml) (R & D Systems, Minneapolis, Minn.) applied.Following additional incubation and rinses, biotinylated anti-goatIgG/avidin D horseradish peroxidase was added. Binding was detected bydevelopment with ABTS [2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid)]; absorbance was read at 405 nm.

EGF—Culture supernatant (200 λ) was pipetted into wells precoated withmonoclonal anti-EGF antibody, according to the manufacturer'sinstructions (R & D Systems). Following incubation and rinses,HRP-linked polyclonal anti-EGF was added and binding detected bydevelopment with tetramethylbenzidine (TMB) substrate; absorbance wasread at 450 nm.

Linear absorbance vs. concentration curves were prepared from resultswith known concentrations of EGF or HB-EGF, and sample concentrationswere determined by plotting absorbance values.

Enzymatic Cleavage

Partially purified APF was incubated with any of threepositionally-specific neuraminidases [2-3; 2-3, 6; 2-3, 6, 8, 9] (allfrom Sigma) at 37° C. for 2 hours; control APF was incubated under thesame conditions but without enzyme. APF was then purified from enzyme byultrafiltration using a 3000 MW cut-off Centricon filter (Amicon,Bedford, Mass.), and antiproliferative activity and lectin-binding ofeach ultrafiltrate determined.

Lectin Binding

HPLC purified APF or enzymatically-treated APF samples were applied tothe following agarose-conjugated lectins and eluted according to themanufacturer's instructions: wheat germ agglutinin, concanavalin A,lentil lectin (all from Amersham), Vicia villosa (Sigma), Erythrinacristagalli, Griffonia simplicifolia I and II, peanut agglutinin,Jacalin lectin (all from Vector Labs, Burlingame, Calif.), orTritrichomonas mobilensis lectin (Calbiochem-Novobiochem Corp., SanDiego, Calif.). Eluates were tested for antiproliferative activity bythe ³H-thymidine incorporation assay.

Sucrose Density Gradient Isoelectric Focusing

HPLC-purified APF was fractionated by high-speed electrofocusing in a pH2 to 10 sucrose density gradient formed at 15 W for 18 hours with an LKB8100-1 column (LKB Instruments Inc., Gaithersburg, Md.). The pH of thefractions was determined at 40° C., and antiproliferative activity ofthe neutralized (pH 7.0) fractions was determined in normal bladdercells using the ³H-thymidine incorporation assay.

APF Synthesis

The synthesis of the peptides was carried out by solid phase methods onthe Nautilus 2400 synthesizer (Argonaut Technologies, Foster City,Calif.) utilizing standard Fmoc chemistry on alanyl 2-chlorotrityl resin(Calbiochem-Novobiochem). Fmoc-protected amino acids (Anaspec Inc., SanJose, Calif.) were coupled utilizing N-{(dimethylamino)-1H-1, 2,3-triazolo[4, 5-b]pyridin-1-ylmethylene}-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU) (Sigma-Aldrich, Milwaukee, Wis.) and1-hydroxy-7-azabenzotriazole (HOAt) (Anaspec, Inc.) reagents. All otherreagents were purchased from Sigma-Aldrich. All intermediates and thefinal products were verified by mass spectrometry.

Fmoc protected O-α-(N-acetyllactosamine)-L-Threonine. Fmoc-L-Thr(Calbiochem-Novabiochem) was converted to phenacyl ester andglycosylated with 2-azido-1-α-bromo-hexa-O-acetyl-2-deoxylactose in thepresence of silver triflate according to a slight modification of theprocedure by Leuck and Kunz (Leuck and Kunz, 1997). The reaction wascarried out a −40° C. that ensured >98% selectivity for the α-anomer.The anomeric purity was determined by proton NMR spectroscopy. Thephenacyl ester was de-protected by zinc/acetic acid/acetic anhydride,which also resulted in the simultaneous reduction of the azido group andacetylation of the resulting amino group (Svarovsky and Barchi, 2003).The final product was purified by preparative, reverse phase (C8 column)HPLC.

Fmoc protected O-β-(N-acetyllactosamine)-L-Threonine. The procedure forproduction of the β anomer was identical to that for production of the αanomer except that the glycosylation of threonine by2-azido-1-α-bromo-hexa-O-acetyl-2-deoxylactose was carried out at −20°C. The product generated by this procedure was a mixture of the α (90%)and β (10%) anomers, which were readily separated by silica gel flashchromatography using an ethyl acetate/hexanes gradient.

Fmoc protected O-α-[Galβ(1→3)GalNAc]-L-Threonine. The syntheticprocedure was similar to the method used to produce the Fmoc protectedO-α-(N-acetyllactosamine)-L-Threonine. Fmoc-L-Threonine phenacyl esterwas glycosylated by the trichloroacetimidate-disaccharide donor in thepresence of boron trifluoride diethyl etherate, following the procedurepublished by Qiu et al. (Qiu et al., 1996) with slight modifications.The conversion of the azido group and the deprotection of the phenacylester were identical to the procedures used in the Fmoc protectedO-β-(N-acetyllactosamine)-L-Threonine synthesis.

General method for glycopeptide synthesis. The glycosylatedFmoc-protected threonine was activated by HATU/HOAt and added to thegrowing peptide chain in presence of Hunig's base for a prolongedcoupling time (16 hours). The glycopeptide was cleaved from the resinwith a mixture of trifluoroacetic acid, water, tri-isopropylsilane(90:5:5 v/v/v), the solvent was removed in vacuo, and the residue wasdried under high vacuum. The crude, dry glycopeptide was dissolved inanhydrous methanol and treated with sodium methoxide powder for 30 min.When HPLC-MS indicated the complete removal of the acetyl groups, thereaction was stopped with acetic acid and evaporated to dryness. Thecrude deacetylated product was purified by preparative HPLC using a C8reverse phase column.

Sialylation of N-terminal threonine hexosamide residue. TheN-acetylhexosamine derivatives of the peptides were sialylatedenzymatically using recombinant rat α-2,3 (N) sialyltransferase (EMDBiosciences, Inc., La Jolla, Calif.) and CMP-N-acetyl neuraminic acidsubstrate (Sigma) in 250 mM MOPS buffer pH 7.4. All crude glycopeptideswere purified by reverse phase HPLC on a C8 column, and the purifiedpeptides were analyzed by mass spectrometry.

Northern Blot Analysis

Total RNA was extracted from explanted bladder epithelial cells from 6patients with interstitial cystitis and their age-, race-, andgender-matched asymptomatic controls using Trizol/chloroform (Gibco-BRL)extraction. Equivalent amounts of RNA from each sample were loaded ontoa 1% agarose/2% formaldehyde gel, separated by standard gelelectrophoresis and transferred to a nylon membrane. DIG-labeled probefor APF mRNA was prepared by random labeling using the known sequence ofnucleotides 1626-1632 (accgtgcccgccgcggtggtggtcgcc) (SEQ ID NO:2) ofhuman frizzled 8 protein (EMBL NM_(—)031866; SEQ ID NO:7, for example)and the DIG Northern Starter Kit with SP6/T7/T3 RNA polymerase (RocheApplied Science, Indianapolis, Ind.); DIG-labeled probe for beta actinmRNA was purchased from Roche Applied Science (prepared usingnucleotides 69-618 of human beta actin) (EMBL HSAC07). Blots weredeveloped by chemiluminescence detection with CDP-Star (Roche AppliedScience).

Anti-APF Antibodies

Synthetic APF peptide was generated by preparing an APF compositioncomprising a peptide moiety comprised of a derivative of SEQ ID NO:1, inwhich an N-terminal cysteine residue was added (SEQ ID NO:6). Thepeptide was coupled to KLH at the cysteine residue (serving as thelinking amino acid) via the sulfur atom, and injected (0.5 mgpeptide/rabbit) into two New Zealand White rabbits for antibodyproduction. Following two monthly booster injections, serum washarvested, immunoglobulin fraction purified, and its reactivity againstHPLC-purified native APF and KLH tested in a dot-blot format, usingHRP-conjugated goat anti-rabbit IgG for the secondary antibody. As shownin FIG. 7, these antibodies detected APF and KLH, and a standard curveagainst known varying concentrations of APF could be generated.Preimmune antibodies did not bind to either APF or KLH.

EXAMPLE 2 Identification of Amino Acids by Mass Spectrometric Ananlysis

In specific aspects of the invention, an APF molecule is identifiedand/or characterized by any suitable means in the art, such as throughthe characterization of the peptide, sugar, or glycopeptide moiety ofAPF. In one specific embodiment, mass spectrometry is utilized. In otherembodiments, techniques such as nuclear magnetic resonance or proteomictechniques (including isotope-coded affinity assays), and sensitivechromatographic methods may be utilized, for example.

Microcapillary reversed-phase liquid chromatography was used to obtainextremely pure preparations of APF peptide for mass spectrometry.Analysis of three preparations of HPLC-purified APF using themicrocapillary technique indicated the presence of three peptide peaksin approximately equal proportions in each preparation, only one ofwhich had antiproliferative activity against primary bladder epithelialcells in vitro. Ion trap mass spectrometric analysis of the active peakindicated a molecular weight of 1482.8 Daltons (FIG. 1A). Analysis ofpeaks generated by collision-induced dissociation of the activesubstance indicated a terminal sialic acid linked to a hexose moiety,which in turn was linked to an N-acetylhexose moiety (FIG. 1B);detectable amino acid moieties present from the N terminus followingfurther dissociation of the 827 Dalton peptide moiety includedalanine-alanine-valine-valine-valine-alanine (FIG. 1C). The remainingN-terminal amino acids present had a combined molecular weight of 351.4Daltons. Search for a gene encoding a homologous peptide indicated 100%homology between a sequence with the appropriate total molecular weight(T-V-P-A-A-V-V-V-A: SEQ ID NO:1) and amino acids 541-549 in the 6thtransmembrane region of frizzled 8, a Wnt ligand receptor (Qiu et al.,1996).

The peptide moiety of the present invention may also compriseconservative variants of SEQ ID NO:1, such as, for example, a peptidecomprising the amino acid sequence of S-V-P-A-A-V-V-V-A (SEQ ID NO:3);T-V-P-A-A-V-V-L-A (SEQ ID NO:4); S-L-P-A-A-V-V-V-A (SEQ ID NO:5); orC-T-V-P-A-A-V-V-V-A (SEQ ID NO:6). It is contemplated that theseconservative variants include known conservative substitutions oradditions, as in the case of SEQ ID NO:6, defined by chemicalproperties. For example, serine, threonine and cysteine compriseheteroatoms (atoms other than carbon or hydrogen having nucleophilic orelectrophilic properties exploitable as linking means), specificallyoxygen (O) and sulfur (S) which provides a means to link to at least onesugar moiety of the present compositions and are considered conservativevariants when interchanged in the peptides and glycopeptides of thepresent invention. In another example, at least one or more leucines issubstituted for any one of the valine subunits.

The skilled artisan is aware that amino acid substitutions can be madethat do not change substantially the functionality of the peptide and/orpolypeptide. Therefore, the present invention contemplates aglycopeptide comprising a peptide having a sequence essentially as setforth in SEQ ID NO:1. The term “a sequence essentially as set forth inSEQ ID NO:1” means that the sequence substantially corresponds to aportion of SEQ ID NO:1 and has relatively few amino acids which are notidentical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:1. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as aprotein having a sequence essentially as set forth in SEQ ID NO:1 andthat is associated with a diseased state of an epithelial cell.Accordingly, sequences which have less than about 50%, between about 50%and about 65%, 70% and about 80%; or more preferably, between about 85%and about 90%; or even more preferably, between about 90 and 95% andabout 99%; of amino acids which are identical or functionally equivalentto the amino acids of SEQ ID NO:1 will be sequences which are“essentially as set forth in SEQ ID NO:1”. However, in specificembodiments, the APF molecule still comprises activity by substitutingmore than one amino acid and may still be active. For example, theentire peptide may be substituted with derivatized amino acids orpeptidomimetic agents, which may be hydrophobic, or another hydrophobicmoiety, such as a lipid.

Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention or DNA segments which encode them and still obtaina functional molecule that encodes a protein or peptide with desirablecharacteristics. The following is a discussion based upon changing theamino acids of a protein to create an equivalent, or even an improved,second-generation molecule. The amino acid changes may be achieved bychanging the codons of the RNA sequence, according to the followingcodon table: TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and messenger RNA sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the disclosed compositions, or corresponding DNA orRNA sequences which encode said peptides without appreciable loss oftheir biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within.+−0.2 is preferred, those which are within .+−0.1 are particularlypreferred, and those within .+−0.05 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

It will also be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids andyet still be essentially as set forth in one of the sequences disclosedherein, so long as the sequence meets the criteria set forth above,including the maintenance of biological protein activity where proteinexpression is concerned. The addition of terminal sequences particularlyapplies to sequences, which may, for example, include variousunnaturally occurring amino acid sequences flanking either of the N-orC- termini to allow for facile covalent linkage to another molecule, i.e., a sugar residue.

EXAMPLE 3 Identification of Sugar Moieties by Lectin Binding Analysis

In specific aspects of the invention, an APF molecule is identifiedand/or characterized by any suitable means in the art. In one specificembodiment, lectin binding analysis is utilized to identify sugarmoieties. Other methods for sugar identification including, but notlimited to, chemical degradation, NMR spectroscopy, mass spectrometry,and antibody binding can also be used to identify sugar moieties.

To determine the identity and linkage of the hexose and hexosaminemoieties, HPLC-purified APF was incubated in its native state withvarious agarose-conjugated lectins and the eluates tested forantiproliferative activity (Table 2). TABLE 2 Lectin Binding AnalysisAPF Binding Lectin Reported Specificity Native Neuraminidase-treatedWheat germ agglutinin sialic acid; terminal GlcNAc + ND Tritrichomonasmobilensis sialic acid + ND Peanut agglutinin Galβ1-3GalNAc; Galactose− + Concanavalin A Mannose, Glucose − − Lentil lectin Mannose, Glucose;terminal GlcNAc − − Vicia villosa Terminal GalNAc; Galα1-3GalNAc − −Griffonia simplicifolia I Galactose; GalNAc − + Griffonia simplicifoliaII Terminal GlcNAc − − Jacalin GalNAc; Galactose − + Erythrinacristagalli Galβ1-4GlcNAc; Galactose − +

Native APF bound to wheat germ agglutinin and Tritrichomonas mobilensislectins, but not to a variety of other lectins, confirming the likelypresence of a terminal sialic acid residue. Treatment of native APF witha neuraminidase that cleaves sialic acid linked by any of four knownlinkages [2,3; 2,6; 2,8; or 2,9] did not decrease its biologicalactivity, but did allow subsequent binding of APF to Griffoniasimplicifolia I, peanut agglutinin, Jacalin lectin and Erythrinacristagalli with elution of biologically active toxin. These resultsindicate the possible presence of galactose and N-acetyllactosamine(galactose β1-4 N-acetylglucosamine), respectively. Apparent lack ofbinding of the desialylated APF to Vicia villosa lectin indicated thatthe disaccharide moiety remaining did not consist ofgalactosα1-3N-acetylgalactosamine, and lack of binding to Griffoniasimplicifolia II confirmed that the N-acetylglucosamine moiety was notterminal following removal of sialic acid. The sialic acid wassubsequently proven to be linked via a 2,3 bond to the galactose moietyby demonstration of binding to Griffonia simplicifolia I and Erythrinacristagalli lectins after digestion with neuraminidase specific for 2,3bond cleavage. The deduced structure of APF was therefore sialic acid2,3 linked to either galactose β1-4 N-acetylglucosamine or galactoseβ1-3 N-acetylgalactosamine, which was in turn 0-linked to threonine onthe N-terminus of the peptide. Isoelectric focusing of the completesialylated native APF indicated an acidic peptide with an isoelectricpoint of approximately 2.3.

EXAMPLE 4 Total Synthesis of APF

The invention encompasses any APF molecule having antiproliferativeactivity, particularly for bladder cells, comprising one or more sugarmoieties and a hydrophobic moiety. Isolated natural APF molecules areencompassed in the invention, as are synthetic APF molecules. Inparticular, an APF molecule is synthesized by any suitable means in theart. In specific embodiments, however, the molecule is synthesized asdescribed in this Example.

Because APF bound to Erythrina cristagalli, and because it is a secretedrather than membrane-bound peptide, synthesis started with aN-acetylglucosamine (GlcNAc) linked to threonine using both α andβ-glycosylated amino acid building blocks for solid phase synthesis ofthe nonaglycopeptide. A chemoenzymatic approach was taken to constructthe complete molecule via synthesis of appropriately Fmoc-protectedN-acetyllactosamine-threonine glycoamino acids in both configurations.These were then incorporated into a peptide chain containing the othereight amino acids. Because it was known from the lectin binding studiesthat the sialic acid moiety was not necessary for APF activity, both αand β-N-acetyllactosamine modified peptides were then assayed for theirantiproliferative activity as well as their ability to regulate specificgrowth factor production by explanted primary normal bladder epithelialcells.

Like native APF, the nonsialylated alpha anomer of theN-acetyllactosamine derivative was a potent inhibitor of cellproliferation, having an IC₅₀ of approximately 0.4 nM; maximalinhibition was possible using as little as 1 nM of the synthetic toxin(FIG. 2). In comparison, the beta anomer of the N-acetyllactosaminederivative and the nonglycosylated peptide had less measurable activity.The neuraminic acid unit was therefore then added to the alpha anomerusing recombinant 2,3-sialyltransferase, and the antiproliferativeactivity of the sialylated derivative determined to be similar to thenonsialylated glycopeptide (FIG. 2). The same 1 nM concentration ofsialylated or nonsialylated alpha anomeric synthetic APF that maximallyinhibited 3H-thymidine incorporation was also shown to significantlydecrease cell counts (data not shown), as well as significantly decreaseHB-EGF production and increase EGF production by primary bladder cells(Table 3). APF as determined by all of these analyses was therefore analpha anomeric sialoglycopeptide. TABLE 3 Effect of Native APF vs.Synthetic APF and its Derivatives on Bladder Epithelial Cell GrowthFactor Production HB-EGF (ng/ml) EGF (ng/ml) Native APF 0.18 ± 0.25* 0.49 ± 0.14* Mock APF 5.24 ± 1.8  0.02 ± 0.02 Synth APF (hexNAc/sial)0.13 ± 0.1^(†)  0.54 ± 0.12^(†) Synth APF (hexNAc) 0.05 ± 0.1^(†)  0.38± 0.01^(†) Synth APF (peptide alone) 4.65 ± 0.5 0.001 ± 0.002*p < 0.0001 compared to Mock APF^(†)p < 0.0001 compared to Peptide alone

However, while a beta anomeric form of N-acetylglucosamine linked tothreonine has been previously described in eucaryotic cells, the alphaanomeric form has not, whereas O-α-N-acetylgalactosamine is commonlyfound in modified eucaryotic cell proteins. Therefore APF wassynthesized to include the alpha anomeric form of N-acetylgalactosaminelinked β→3 to galactose. As also shown in FIG. 2, this synthetic APF hadactivity similar to the synthetic compound comprising an alphaN-acetylglucosamine. Microcapillary LC-MS/MS in which desialylatednative APF was spiked with either the GalNAc-containing orGlcNAc-containing synthetic glycopeptide (FIGS. 3A and 3B) was thereforeused to establish the correct structure for APF. Both the native andsynthetic GalNAc-containing APF derivatives had an identical retentiontime that was readily distinguishable from that of the GlcNAc-containingcompound. The correct structure of APF is therefore theGalNAc-containing sialoglycopeptide shown in FIG. 4.

EXAMPLE 5 Inhiition of Bladder Cancer Cell Proliferation by Native andSynthetic APF

Additional evidence that the synthetic and native APF were the same wasprovided by measurement of their antiproliferative activities against abladder carcinoma T24 cell line, using live cell count as determined bytrypan blue exclusion. As shown in FIG. 5, these cells were sensitive toboth native and synthetic APF species at approximately the sameconcentration.

EXAMPLE 6 Identification of APF MRNA by Northern Blot Analysis

Northern blot analysis of mRNA extracted from the explanted bladderepithelial cells of 6 IC patients and 6 normal controls was performed toprovide additional evidence that APF was a frizzled 8-related peptide.As shown in FIG. 6, a small (approximately 450 b.p.) mRNA species thatbound to a probe encoding the nonapeptide was present only in theextracts of all 6 cell explants from patients with IC previously shownto produce APF, but not in extracts of any explanted cells from age-,race- and gender-matched controls that did not produce APF. Although afaint band was also seen at approximately 3100 b.p. in IC but notcontrol specimens on three of five experiments, it was not detectable inthe other two experiments, and the predominant band was at 450 b.p. eachtime. However, no band was seen at the size of full length mRNA forhuman frizzled 8 (4 Kb), and no bands were seen for control cells in anyexperiment. In comparison, all cells from both groups appeared toproduce similar amounts of beta actin mRNA.

EXAMPLE 7 Therapeutic Embodiments with Inhibitory Compounds for APF

In some aspects of the invention, it is beneficial to provide to anindividual in need thereof an inhibitory compound for APF, therebyreducing partly or completely the anti-cell proliferation activity orproduction of APF. For example, for treating a bladder condition aninhibitor of APF is delivered to an individual suffering from at leastone symptom of the bladder condition or to an individual suspected ofhaving the bladder condition. In specific embodiments, the inhibitor ofAPF is an antibody that binds thereto, or a small molecule inhibitor ofAPF binding to its target. In other aspects, the inhibitor of APF is acompound that inhibits APF production, such as an antisenseoligonucleotide or small interfering RNA, or any compound that couldalso inhibit APF production. In another aspect, the inhibitor of APF isa compound that stimulates the breakdown of APF.

The inhibitor of APF may be delivered to the individual by any suitablemeans. In specific embodiments of the present invention, the inhibitorof APF is comprised as an oral medication and/or is delivered via acatheter. A sufficient amount may be delivered directly to bladdertissue or it may be delivered systemically. A sufficient amount is onethat ameliorates at least one symptom or finding (sign) of the bladdercondition, and a skilled artisan recognizes standard methods todetermine such an amount.

EXAMPLE 8 Therapeutic Embodiments with APF

In some aspects of the invention, it is beneficial to deliver to anindividual in need thereof an APF composition to provide its anti-cellproliferation activity. For example, for treating bladder cancer an APFcomposition is delivered to an individual suffering from at least onesymptom or finding (sign) of bladder cancer or to an individualsuspected of having bladder cancer. FIG. 8 demonstratesantiproliferative activity against normal human bladder epithelial cellsof 2 different peptides (#1, corresponds to SEQ ID NO:1, and #4,corresponds to SEQ ID NO:5) and their sugar derivatives, having two (#2and #5, each unsialyated) and three (#3 and #6) sugar moieties.

In other embodiments, prostate cancer cells (such as the exemplary LNCaPcells) are treated with APF compositions. LNCaP cells were plated at2×10⁴ cells per well of a 24 well tissue culture plate in DMEM mediumcontaining 10% fetal bovine serum, 1% L-glutamine, 1%antibiotic/antimycotic solution, and grown at 37° C. in a 5% CO₂atmosphere. The next day the medium was changed to DMEM containing thesame additives except without fetal bovine serum, after whichHPLC-purified APF or an equivalent amount of mock APF was added to eachwell. Live cell counts were performed on Day 3 of incubation by trypanblue exclusion. Values are the percent decrease in cell count comparedto cell control given medium alone, and are given as the mean oftriplicate wells; vertical lines are the standard deviation. The cellswere sensitive to the antiproliferative activity of native purified APF(FIG. 9).

The APF composition may be delivered to the individual by any suitablemeans. In specific embodiments of the present invention, the APFcomposition is comprised as an oral medication and/or is delivered via acatheter, orally, intravenously, topically, subcutaneously,transcutaneously, intramuscularly, intra-jointly, parenterally,peritoneally, intranasally, intravesically, or by inhalation. Asufficient amount may be delivered directly to bladder tissue or it maybe delivered systemically. A sufficient amount is one that ameliorateswhen given alone or in combination with other agents or other types oftherapy at least one symptom or objective finding of the bladder cancer,and a skilled artisan recognizes standard methods to determine such anamount.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

PATENTS

-   U.S. Pat. No. 5,811,393-   U.S. Pat. No. 5,916,871-   U.S. Pat. No. 5,962,645-   U.S. Pat. No. 6,156,522-   U.S. Pat. No. 6,232,289-   U.S. Pat. No. 6,376,197-   U.S. Pat. No. 6,600,018

PUBLICATIONS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1-75. (canceled)
 76. A method of treating an individual for cancer,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising a urinary bladderantiproliferative factor having one or more sugar moieties, wherein atleast one sugar moiety is linked to a hydrophobic moiety.
 77. The methodof claim 76, wherein the cancer comprises an epithelial cancer.
 78. Themethod of claim 76, wherein the cancer comprises bladder cancer.
 79. Themethod of claim 76, wherein the cancer comprises prostate cancer. 80-84.(canceled)
 85. A method of treating a hyperplasia, comprising the stepof administering a therapeutically effective amount of a compositioncomprising a urinary bladder antiproliferative factor having one or moresugar moieties, wherein at least one sugar moiety is linked to ahydrophobic moiety.
 86. A method of enhancing cancer treatment of anindividual, comprising administering to the individual a therapeuticallyeffective amount of a composition comprising a urinary bladderantiproliferative factor having one or more sugar moieties, wherein atleast one sugar moiety is linked to a hydrophobic moiety.
 87. The methodof claim 86, wherein administration of the composition enhanceschemotherapy, radiotherapy, immunotherapy, surgery, gene therapy, or acombination thereof.
 88. The method of claim 86, wherein the compositionis administered prior to the cancer treatment being enhanced,concomitant with the cancer treatment being enhanced, subsequent to thecancer treatment being enhanced, or a combination thereof.